Methods for improving traits in plants

ABSTRACT

The present invention discloses a method for screening for and identifying a desirable plant improving trait, said method comprises steps of: (a) obtaining genetic material from a sampling of a predefined source and (b) constructing an expression library from said genetic material. The aforementioned method further comprises steps of: (c) producing plants transformed with said expression library at a transformation efficiency of at least 0.05%-30%, representing at least 102-1010 transgenes; (d) screening for transformed plants expressing said desirable trait; and (e) identifying said transgene of said transformed plants expressing said desirable trait.

RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.16/496,445 filed on Sep. 22, 2019, which is a National Phase of PCTPatent Application No. PCT/IL2018/050349 having International filingdate of Mar. 27, 2018, which claims the benefit of priority of U.S.Provisional Application Nos. 62/477,517 and 62/644,600, filed on Mar.28, 2017 and Mar. 19, 2018, respectively. The contents of the aboveapplications are all incorporated by reference as if fully set forthherein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII txt. format and is hereby incorporatedby reference in its entirety. Said ASCII copy, is named seq.listing1754-P-01-PCT_ST25 and is 491 KB in size.

FIELD OF THE INVENTION

The present invention generally relates to the field of improving traitsin plants. More particularly, the present invention relates to improvingtraits in plants by transformation of expression libraries frompredefined sources into plants and screening for desirable traits.

BACKGROUND OF THE INVENTION

The world population is estimated to be 9.2 billion in 2050. Tosufficiently feed this population, the total food production will haveto increase by 60%-70%. Climate models predict that warmer temperaturesand increases in the frequency and duration of drought during thepresent century will have negative impact on agricultural productivity.For example, maize production in Africa could be at risk of significantyield losses as researchers predict that each degree-day that the cropspends above 30° C. reduces yields by 1% if the plants receivesufficient water. These predictions are similar to those reported formaize yield in the United States. It has been further shown that maizeyields in Africa decreased by 1.7% for each degree-day the crop spent attemperatures of over 30° C. under drought. Wheat production in Russiadecreased by almost one-third in 2010, largely due to the summer heatwave. Similarly, wheat production declined significantly in China andIndia in 2010, largely due to drought and sudden rise in temperaturerespectively, thereby causing forced maturity. These new globalchallenges require a more complex integrated agriculture.

In addition global warming leads to the concurrence of a number ofabiotic and biotic stresses, thus affecting agricultural productivity.Occurrence of abiotic stresses can alter plant-pest interactions byenhancing host plant susceptibility to pathogenic organisms, insects,and by reducing competitive ability with weeds. On the contrary, somepests may alter plant response to abiotic stress factors.

Biotic stress factors are caused by pathogens, insects, pests, weeds, orintraspecific competition for resources. The ability of biotic stressfactors to cause yield or quality loss depends on the environment andthus may vary from region to region or from one agroecology to another.For example, in Australia, barley foliar diseases are some of the majorbiotic stress factors causing substantial yield and quality losses.Although it is known that some plant species have resistance to variousdiseases, they are hard or even impossible to breed in conventionalmethods.

The challenge is to create crops that are resistance to biotic stressfactors and are flexible and adaptable to diverse environments andpopulations. There are currently two major acceptable ways to adaptcrops to new environments: developing new crops through conventionalbreeding (long-term endeavor starting with domestication) andintroducing target traits into existing crops through plant breeding,which includes genetic engineering. To maintain productivity in the faceof increased climatic variability, both the population and the plantcultivars will need to be continually developed to withstand “new”climate extremes and other stresses such as diseases, pathogens,insects, pests etc. In addition there is a constant need to find newherbicide tolerance or resistant genes for new chemicals and newherbicides mode of action.

Genetic engineering has the potential to address some of the mostchallenging biotic and abiotic constraints faced by farmers, which arenot easily addressed through conventional plant breeding alone.

Advantageous outcomes of these genetic modifications include increasedfood production, reliability, and yields; enhanced taste and nutritionalvalue; and decreased losses due to various biotic and abiotic stresses,such as fungal and bacterial pathogens. These objectives continue tomotivate modern breeders and food scientists, who are seeking for newergenetic modification methods for identifying, selecting, and analyzingindividual organisms that possess genetically enhanced features.

The option to transform plants with foreign genes and/or genes from thesame specie or genus, that are hard or impossible to breed, overcomesspecies barriers, making it possible to exploit powerful ‘super-traits’that are not attainable through traditional methods. However, themolecular interactions and outcomes of introduced trans-genes andendogenous genes are not predictable.

When genes coding for certain traits are transferred, typically from oneplant species to another, the desired traits are not always expressedunless the environment interacts with the genes in the anticipated waytriggering the desired response, which depends on the regulatingsequences inserted with the gene. This means that new transgeniccultivars, developed under laboratory conditions in a controlledclimate, have to be tested under field conditions, as in moretraditional breeding methods, so currently there is little difference inthe speed with which either method will result in the release of newcultivars.

The knowledge gained from basic plant research will underpin future cropimprovements, but effective mechanisms for the rapid and effectivetranslation of research discoveries into public good agriculture remainto be developed.

U.S. Pat. Nos. 6,030,779 and 6,368,798 disclose a process foridentifying clones having a specified enzyme activity by selectivelyisolating target nucleic acid from genomic DNA population, by use ofpolynucleotide probe identifying the nucleic acid sequence encoding anenzyme having the specified enzyme activity; and transforming a hostwith the isolated target nucleic acid to produce a library of cloneswhich are screened for the specified enzyme activity.

U.S. Pat. No. 6,972,183 discloses a process for screening an expressionlibrary to identify clones expressing enzymes having a desired activity.The process involves generating from genomic DNA samples of one or moremicroorganisms an expression library comprising a plurality ofrecombinant cell clones, and then introducing into capillaries in acapillary array a substrate and a subset of the clones. Interaction ofthe substrate and a clone expressing an enzyme having the desiredactivity produces an optically detectable signal, which can then bespatially detected to identify capillaries containing clones producingsuch a signal. The signal-producing clones can then be recovered fromthe identified capillaries.

EP patent application 1025262 and US patent application 20020150949teach a process for identifying clones having a specified activity ofinterest, by (i) generating expression libraries derived from nucleicacid directly isolated from the environment; (ii) exposing saidlibraries to a particular substrate or substrates of interest; and (iii)screening said exposed libraries utilizing a fluorescence activated cellsorter to identify clones which react with the substrate or substrates.

US patent application 20100152051 relates to a method for theidentification and/or characterization of clones conferring a desiredbiological property from an expression library. The method comprises thestep of screening for the expression of at least one (poly)peptide, suchas a tag expressed as a fusion protein, together with a recombinantinsert of a clone of said expression library. Said (poly)peptide may befused N-terminally or C-terminally to said insert. The method furthercomprises the steps of contacting a ligand specifically interacting withthe (poly)peptide expressed by the insert of a clone conferring saiddesired biological property.

All the above methods are based upon screening a DNA library (producedfrom microorganisms or environmental sample) for a specific sequence orbiochemical activity via interaction with a predetermined probe. Inaddition, the screening and selection for a clone having thepredetermined sequence or activity is performed prior to transformationinto plant cells and could be expressed in plant cells (tissue cultures)but not in whole plants. Thus by the up-to-date used methods, only thepreselected clone is expressed in plants and the expression and effectof the selected sequence in plants is unpredictable. In addition, in themethods described above, one can screen only for known activities basedon prior knowledge. Thus, these methods are limited under the scope ofknown enzyme activities and enzyme families and prior known function.

In view of the above, there is a long felt need for efficient methodsfor screening and identifying unknown sequences conferring desirableplant improving traits.

SUMMARY OF THE INVENTION

It is therefore one object of the present invention to disclose a methodfor screening for and identifying a desirable plant improving trait, themethod comprises steps of: (a) obtaining genetic material from asampling of a predefined source; (b) constructing an expression libraryfrom said genetic material; wherein said method further comprises stepsof: (c) producing plants transformed with said expression library at atransformation efficiency of at least 0.05%-30%, representing at least102-1010 transgenes; (d) screening for transformed plants expressingsaid desirable trait; and (e) identifying said transgene of saidtransformed plants expressing said desirable trait.

It is a further object of the present invention to disclose the methodas defined above, further comprising a step of editing a target gene ina desirable crop plant according to genetic information obtained fromsaid transgene.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said editing of said target geneis performed using any genome editing system or method including systemsusing engineered nucleases selected from the group consisting of:meganucleases, zinc finger nucleases (ZFNs), transcriptionactivator-like effector-based nucleases (TALEN), clustered regularlyinterspaced short palindromic repeats (CRISPR) system and anycombination thereof.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said predefined source comprisesplant, microbial, fungal or other organisms or parts thereof of anenvironmental niche.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said screening step comprisesmeasurements of said transformed plants as compared to control plants,said measurements are selected from the group consisting of: turgorpressure measurements, plant death, leaf area, plant shoots freshweight, leaf number, branch fresh weight, main branch length, flowersyield, pods or fruits yield, chlorosis, damage to leaves, state orperformance of plants and any combination thereof.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said control plant is a plant ofthe same genus as said transgenic plant and lacking said transgene or aplant of the same genus as said transgenic plant, lacking said transgeneand transformed with a known gene conferring said plant improving trait.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said step (a) further comprisessteps of enriching said genetic material by growth on rich media or onselective media.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said step (a) further comprisessteps of enhancing expression of said desirable trait by culturing saidgenetic material on selective media for said desirable trait.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said step (b) comprises steps ofproducing prokaryotic cDNA library or eukaryotic cDNA library or both.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said step (b) further comprisessteps of cloning said cDNA library into at least one binary vector.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said binary vector comprises aconstitutive promoter or a stress induced promoter.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said binary vector comprisesbacterial selection marker and plant transformation selection marker.

It is a further object of the present invention to disclose the methodas defined in any of the above, further comprises steps of transformingsaid cloned binary vectors into host cells.

It is a further object of the present invention to disclose the methodas defined in any of the above further comprises steps of transformingsaid cloned binary vectors into Agrobacterium tumefaciens.

It is a further object of the present invention to disclose the methodas defined in any of the above further comprises steps of introducingsaid transformed Agrobacterium tumefaciens into at least one of: wholeplant, plant tissue and plant cell.

It is a further object of the present invention to disclose the methodas defined in any of the above, comprises steps of introducing saidtransformed Agrobacterium tumefaciens by spraying said plants with aninoculum comprising transformed Agrobacterium.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said step (d) comprises growingsaid transformed plants under conditions selective for said desirabletrait.

It is a further object of the present invention to disclose the methodas defined in any of the above, further comprises steps of: (f)collecting T1 seeds from said transformed plants of step (d); (g)determining seed library transformation efficiency of said T1 seeds; (h)sowing said T1 seeds of step (e) under selective conditions allowingscreening and selection of transformed plants expressing said desirabletrait; (i) testing said selected plants expressing said desirable traitof step (g) for presence of said transgene; and (j) isolating andsequencing said transgene of said selected transformed plants positivelytested for said transgene of step (h).

It is a further object of the present invention to disclose the methodas defined in any of the above, further comprises steps of (k)collecting T2 seeds from said plants of (h), which are found positivefor presence of said transgene; (l) growing plants of said T2 seedsunder selective conditions allowing screening and selection oftransformed plants expressing said desirable trait as compared tocontrol plants transformed with known genes conferring said desirabletrait; and (m) optionally, isolating and sequencing said transgene ofsaid selected plants of step (j).

It is a further object of the present invention to disclose the methodas defined in any of the above, comprises steps of (a) recloning andsequencing said isolated transgene of step (i) and/or (l); (b)transforming said recloned transgene into plants; (c) screening saidtransformed plants of step (b) for selection of transformed plantsexpressing said desirable trait; (d) isolating said transgene from saidselected plants of step (c); and (e) optionally, repeating steps (a) to(d).

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said environmental nichecomprises ecological niche, populations, habitats, gene pools,prokaryotic culture, eukaryotic culture and any combination thereof.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said environmental nichecomprises microbiome, microbiota, microbial culture, plant, yeast,algae, nematode or any other organism or combinations thereof.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said environmental nichecomprises predefined biotic factors, abiotic factors and a combinationthereof.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said sampling comprises soilsample, water sample, organic matter sample and any combination thereof.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said desirable trait is selectedfrom the group consisting of resistance or tolerance to at least onebiotic stress, resistance or tolerance to at least one abiotic stress,improved yield, improved biomass, improved food qualities and values,improved grain yield, herbicide or chemical resistance or tolerance andany combination thereof.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said abiotic stress is selectedfrom the group consisting of: drought, salinity, heat, cold, fertilizeruptake, fertilizer usage efficiency and any combination thereof.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said biotic stress is selectedfrom the group consisting of: plant diseases, pathogens, bacteria,viruses, fungi, parasites, beneficial and harmful insects, weeds, andcultivated or native plants or any combination thereof.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said step (a) comprises steps ofextracting RNA from said sampling of said predefined environmentalniche.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said RNA extraction is performedaccording to standard commercial kits or according to any other protocolfor extraction of RNA from environmental sampling.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said protocol for extraction ofRNA from environmental sampling comprises steps of: (a) obtaining a soilsample; (b) mixing said soil sample with an extraction buffer comprising500 mM phosphate buffer pH 8 and 5% w/v cetyltrimethylammonium bromide(CTAB) with phenol (pH 8)/chloroform/IAA ratio of 25:24:1; (c)subjecting said mixture of step (b) to 15 min shaking at 37° C. or to abead beater for 1 min; (d) centrifuging said mixture of step (c) at2,500 g for about 10 minutes at room temperature to obtain an aqueousphase; (e) transferring said aqueous phase into a new tube; (f) addingto said aqueous phase within said tube of step (e) an equal amount ofiso-propanol supplemented with 20 mg/ml crystal violet solution toobtain violate stained solution; (g) mixing said solution by invertingsaid tube of step (f) and then incubating said tube for about 30 minutesat room temperature; (h) centrifuging said tube of step (g) at 2,500 gfor about 30 minutes at room temperature to obtain a violet stainedlayer; (i) transferring said violate stained layer into a new tube andcentrifuging said tube for about 5 min at maximal speed to obtain pelletand supernatant; (j) washing said pellet with 80% v/v ice cold ethanoland centrifuging for additional 5 min to obtain pellet and supernatant;(k) removing said supernatant of step (j) and allowing said pellet todry; and (l) suspending said dried pellet in water in a ratio of 100 μlwater to 2 gr of soil of step (a).

It is a further object of the present invention to disclose a plantcomprising said transgene identified by the method as defined in any ofthe above.

It is a further object of the present invention to disclose the plant asdefined above, wherein said plant has at least one plant improving traitas compared to a plant of the same genus lacking said transgene.

It is a further object of the present invention to disclose apolynucleotide sequence obtainable by the method as defined in any ofthe above.

It is a further object of the present invention to disclose thepolynucleotide as defined in any of the above, wherein saidpolynucleotide comprises a nucleotide sequence corresponding to thesequence as set forth in a polynucleotide sequence selected from thegroup consisting of SEQ ID NOs:1 -148 and any combination thereof.

It is a further object of the present invention to disclose apolynucleotide sequence having at least 80% sequence similarity to thepolynucleotide sequence as defined in any of the above.

It is a further object of the present invention to disclose apolypeptide sequence obtainable by the method as defined in any of theabove.

It is a further object of the present invention to disclose thepolypeptide sequence as defined in any of the above, wherein saidpolypeptide comprises an amino acid sequence corresponding to thesequence as set forth in a polypeptide sequence selected from the groupconsisting of SEQ ID NOs: 149-321 and any combination thereof.

It is a further object of the present invention to disclose apolypeptide sequence having at least 60% sequence similarity to thepolypeptide sequence as defined in any of the above.

It is a further object of the present invention to disclose the use ofthe method as defined in any of the above for identifying genesconferring plant improving traits selected from the group consisting ofresistance or tolerance to abiotic stress, resistance or tolerance tobiotic stress, improved yield, improved biomass, improved food qualitiesand values, improved grain yield, herbicide or chemical resistance ortolerance and any combination thereof.

It is a further object of the present invention to disclose the use asdefined in any of the above, wherein said abiotic stress is selectedfrom the group consisting of: drought, salinity, heat, cold, fertilizeutilization and any combination thereof.

It is a further object of the present invention to disclose the use asdefined in any of the above, wherein said biotic stress is selected fromthe group consisting of: plant diseases, pathogens, bacteria, viruses,fungi, parasites, beneficial and harmful insects, weeds, and cultivatedor native plants or any combination thereof.

It is a further object of the present invention to disclose a method forscreening for and identifying a drought or salinity resistance ortolerance improving trait in plants, said method comprises steps of: (a)obtaining genetic material derived from a low moisture or a highsalinity source sample; (b) constructing expression library from saidgenetic material; wherein said method further comprises steps of: (c)producing plants transformed with said expression library at atransformation efficiency of at least 0.5%-30% representing at least10²-10¹⁰ transgenes; (d) screening for transformed plants resistant ortolerant to predetermined drought or salinity conditions; and (e)identifying said transgene of said drought or salinity resistant ortolerant transformed plants of step (d).

It is a further object of the present invention to disclose the methodas defined in any of the above, further comprising a step of editing atarget gene in a desirable crop plant according to genetic informationobtained from said transgene.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said editing of said target geneis performed using any genome editing system or method including systemsusing engineered nucleases selected from the group consisting of:meganucleases, zinc finger nucleases (ZFNs), transcriptionactivator-like effector-based nucleases (TALEN), clustered regularlyinterspaced short palindromic repeats (CRISPR) system and anycombination thereof.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said predefined source comprisesplant, microbial, fungal or other organisms or parts thereof of anenvironmental niche.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said screening step comprisesmeasurements of said transformed plants as compared to control plants,said measurements are selected from the group consisting of: turgorpressure measurements, plant death, leaf area, plant shoots freshweight, leaf number, branch fresh weight, main branch length, flowersyield, pods or fruits yield, chlorosis, damage to leaves, state orperformance of plants and any combination thereof.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said control plant is a plant ofthe same genus as said transgenic plant and lacking said transgene or aplant of the same genus as said transgenic plant, lacking said transgeneand transformed with a known gene conferring said plant improving trait.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said step (b) further comprisessteps of cloning said expression library into at least one binaryvector.

It is a further object of the present invention to disclose the methodas defined in any of the above, further comprises steps of: (f)collecting T1 seeds from said transformed plants of step (c); (g) sowingsaid T1 seeds in soil selective for transformed plants, with watercontent of about 100% capacity; (h) growing plants of said T1 seeds indrought or salinity conditions and/or without irrigation until most ofthe plants die, to produce transformed plants surviving said drought orsalinity conditions; (i) growing said drought or salinity survivingtransformed plants to produce T2 seeds; (j) screening said drought orsalinity surviving transformed plants of step (i) for presence of atransgene; and (k) isolating and sequencing said transgene frompositively screened plants of step (j).

It is a further object of the present invention to disclose the methodas defined in any of the above, further comprises steps of (l)collecting T2 seeds from each of said transgene-containing positivelyscreened drought or salinity surviving transformed plants of step (j);(m) growing T2 plants from each of said transgene-containing T2 seeds ofstep (l) under predetermined drought or salinity conditions as comparedto control plants of the same genus and lacking said transgene ortransformed with known genes conferring drought or salinity tolerance ordrought or salinity resistance; (n) performing drought tolerance orresistance screening measurements for each of said transgene-containingT2 plants as compared to said control plants, said measurements areselected from the group consisting of: turgor pressure measurements,plant death, leaf area, fresh weight, leaf number, branch fresh weight,main branch length, flowers and pods production, Chlorosis and damage toleaves, state or performance of plants and any combination thereof; (o)isolating the transgene from said screened dough or salinity resistanceperforming T2 plants of step (n); (p) optionally, recloning saidtransgene into a binary vector; (q) optionally, transforming said clonedbinary vector into plants and growing said transformed plants underpredetermined drought or salinity conditions; and (r) optionally,repeating steps (l) to (q).

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said step of growing T2 plantscomprises steps of: (a) sowing said T2 seeds in soil selective fortransformed plants, with water content of about 100% capacity; and (b)irrigating said plants when water content in the soil reaches about5-10%.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said predetermined drought orsalinity conditions are selected from the group consisting of lowmoisture, high salinity, dry soil and heat.

It is a further object of the present invention to disclose apolynucleotide sequence obtainable by the method as defined in any ofthe above.

It is a further object of the present invention to disclose thepolynucleotide as defined in any of the above, wherein saidpolynucleotide comprises a nucleotide sequence corresponding to thesequence as set forth in a polynucleotide sequence selected from thegroup consisting of SEQ ID NOs:1 to SEQ ID NO:148 and any combinationthereof.

It is a further object of the present invention to disclose apolynucleotide sequence having at least 80% sequence similarity to thepolynucleotide sequence as defined in any of the above.

It is a further object of the present invention to disclose apolypeptide sequence obtainable by the method as defined in any of theabove.

It is a further object of the present invention to disclose thepolypeptide sequence as defined in any of the above comprises an aminoacid sequence corresponding to the sequence as set forth as set forth inpolypeptide sequence selected from the group consisting of SEQ. ID Nos:149-321 and any combination thereof.

It is a further object of the present invention to disclose apolypeptide sequence having at least 60% sequence similarity with thepolypeptide sequence as defined in any of the above.

It is a further object of the present invention to disclose a method forextracting RNA from a soil sample comprising steps of: (a) obtaining asoil sample; (b) mixing said soil sample with an extraction buffercomprising 500 mM phosphate buffer pH 8 and 5% w/vcetyltrimethylammonium bromide (CTAB) with phenol (pH 8)/chloroform/IAAratio of 25:24:1; (c) subjecting said mixture of step (b) to 15 minshaking at 37° C. or to a bead beater for 1 min; (d) centrifuging saidmixture of step (c) at 2,500 g for about 10 minutes at room temperatureto obtain an aqueous phase; (e) transferring said aqueous phase into anew tube; (f) adding to said aqueous phase within said tube of step (e)an equal amount of iso-propanol supplemented with 20 mg/ml crystalviolet solution to obtain violate stained solution; (g) mixing saidsolution by inverting said tube of step (f) and then incubating saidtube for about 30 minutes at room temperature; (h) centrifuging saidtube of step (g) at 2,500 g for about 30 minutes at room temperature toobtain a violet stained layer; (i) transferring said violate stainedlayer into a new tube and centrifuging said tube for about 5 min atmaximal speed to obtain pellet and supernatant; (j) washing said pelletwith 80% v/v ice cold ethanol and centrifuging for additional 5 min toobtain pellet and supernatant; (k) removing said supernatant of step (j)and allowing said pellet to dry; and (l) suspending said dried pellet inwater in a ratio of 100 μl water to 2 gr of soil of step (a).

It is a further object of the present invention to disclose a method forscreening for and identifying a desirable plant improving trait, saidmethod comprises steps of: (a) obtaining a sampling of a predefinedsource; (b) extracting RNA from said sampling according to the method ofclaim 60; (c) constructing an expression library from said RNA of step(b); wherein said method further comprises steps of: (d) producingplants transformed with said expression library at an efficiency of atleast 0.05%-30% representing at least 10²-10¹⁰ transgenes; (e) screeningfor transformed plants expressing said desirable trait; and (f)identifying said transgene of said transformed plants expressing saiddesirable trait.

It is a further object of the present invention to disclose the methodas defined in any of the above, further comprising a step of editing atarget gene in a desirable crop plant according to genetic informationobtained from said transgene.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said editing of said target geneis performed using any genome editing system or method including systemsusing engineered nucleases selected from the group consisting of:meganucleases, zinc finger nucleases (ZFNs), transcriptionactivator-like effector-based nucleases (TALEN), clustered regularlyinterspaced short palindromic repeats (CRISPR) system and anycombination thereof.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said predefined source comprisesplant, microbial, fungal or other organisms or parts thereof of anenvironmental niche.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said screening step comprisesmeasurements of said transformed plants as compared to control plants,said measurements are selected from the group consisting of: turgorpressure measurements, plant death, leaf area, plant shoots freshweight, leaf number, branch fresh weight, main branch length, flowersyield, pods or fruits yield, chlorosis, damage to leaves, state orperformance of plants and any combination thereof.

It is a further object of the present invention to disclose the methodas defined in any of the above, wherein said control plant is a plant ofthe same genus as said transgenic plant and lacking said transgene or aplant of the same genus as said transgenic plant, lacking said transgeneand transformed with a known gene conferring said plant improving trait.

It is a further object of the present invention to disclose an isolatedpolynucleotide having at least 80% sequence similarity to a nucleotidesequence selected from the group consisting of SEQ ID NOs:1 to SEQ IDNO:148 and any combination thereof.

It is a further object of the present invention to disclose an isolatedpolypeptide having at least 60% sequence similarity to an amino acidsequence selected from the group consisting of SEQ. ID Nos: 149-321 andany combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may beimplemented in practice, several embodiments will now be described, byway of non-limiting example only, with reference to the accompanyingdrawing, in which:

FIGS. 1A-D present schematic illustrations of binary vectors used forinsertion of amplified cDNA clones between the promoter(s) (35S, CBF3,Erd10 and Kin1) and the HSP terminator. FIG. 1A illustrates the pPA-35Hvector, which has a constitutive CaMV 35S promoter. FIGS. 1B-D presentvectors containing stress induced promoters of Arabidopsis thaliana:pPA-CH with CBF3 promoter (FIG. 1B), pPA-EH with Erd10 promoter (FIG.1C) and pPA-KH with Kin1 promoter (FIG. 1D);

FIG. 2 presents a photographic illustration of agrobacterium librarycounting for 3 different libraries on LB petri dishes;

FIGS. 3A-3C presents a photographic illustration of tobacco tissueculture transformed with a library, 7 days after transformation (FIG.3A), 40 days after transformation (FIG. 3B) and 6-8 weeks aftertransformation (FIG. 3C);

FIG. 4 presents a photographic illustration demonstrating selection forphosphinothricin resistance of 10 days old Arabidopsis expressinglibrary seedlings. The green plants are resistant to phosphinothricinwhile small yellow plants are absent of the transgene and thereforesusceptible;

FIG. 5 presents a photographic illustration of T2 and T3 controlledexperiments in the greenhouse;

FIG. 6 presents photographic results of screening for transgenic plantsresistance to drought;

FIG. 7 presents a graphic illustration demonstrating loss of turgorpressure in plants expressing genes used as control relative to soilwater content (dark gray), days after cessation of irrigation;

FIG. 8 presents a graphic illustration showing normalized death scale ofpositive control expressing transgenic plants as compared to GFPexpressing plants;

FIG. 9 graphically shows results of several drought resistance genesidentified by the method of the present invention; and

FIG. 10 graphically shows leaf area analysis of several transgenic plantlines expressing identified novel genes conferring drought resistanceafter re-cloning as compared to positive and negative controls.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description is provided, alongside all chapters of thepresent invention, so that to enable any person skilled in the art tomake use of the invention and sets forth the best modes contemplated bythe inventor of carrying out this invention. Various modifications,however, will remain apparent to those skilled in the art, since thegeneric principles of the present invention have been definedspecifically to provide means and methods for screening and identifyinga desirable plant improving trait.

It is known that some plant species have resistance to various diseases.However, such species are usually hard or impossible to breed inconventional techniques and methods.

The present invention provides a method and platform to discover andidentify genes from plants that have unique and valuable features, suchas disease resistance, abiotic stress resistance or tolerance, foodimproving qualities (e.g. improved oils, protein content, amino acids,vitamins etc.) and then to insert or express them in desired cropsthrough gene editing, or other transformation technique.

It is therefore within the scope of the present invention to introducetarget traits into existing crops through plant breeding, which includesgenetic engineering and gene (genome) editing.

The present invention provides a novel method for screening andidentifying a desirable plant improving trait. The method comprisessteps of: (a) obtaining genetic material from a sampling of a predefinedenvironmental niche or genetic material extracted from other sourcessuch as plants from the same or other genus; and (b) constructing anexpression library from said genetic material. According to coreaspects, the present invention further comprises steps of: (c) producingplants transformed with said expression library at an efficiency of atleast 0.05%-30% representing at least 10²-10¹⁰ transgenes; (d) screeningfor transformed plants expressing said desirable trait; and (e)identifying said transgene of said transformed plants expressing saiddesirable trait.

The present invention provides for the first time a method for screeningfor and selecting unknown sequences derived from predefined sources(e.g. ecological niches and/or plants) which confer improved traits invaluable crop plants. The current method is effective and advantageousupon common and conventional screening methods by the following aspects:

1. An expression library is prepared from genetic material or geneticpool (i.e. RNA) originating from predefined sources, such as extremeenvironment, plant material and other. In this way, only genes which areexpressed in the preselected environmental conditions are used for thescreening procedure in plants.

2. The entire expression library is transformed into plants at anefficiency of 0.05%-30% and representation of at least 10²-10¹⁰ uniquetransgenes.

3. In the method of the present invention, the screening of theexpressed library for the desirable phenotype is performed at the targetorganism, which is the plant. In this way there is no preselection andnew and unique genes for the desired phenotype, which are expressible inplants, are revealed.

In the conventional methods, the first step is selecting genes for apredefined trait in a source genetic material, e.g. by probing a DNAlibrary with known sequences in prokaryotic- or eukaryotic cells, andonly then the preselected gene is expressed in plants. The outcome ofsuch a conventional method is limited and has the following drawbacks:

1. The screening is performed in a host cell/organism which is not thetarget organism (usually in prokaryotic or unicellular organism).

2. The screening is limited since it is performed with known sequencesor probes or activity. It was shown that functional screening methodsrequire detectable levels of enzyme activity that cannot be alwaysachieved, for example, only about 40% of the enzymatic activities arelikely to be detected in E. coli-based expression systems (Gabor et al.,2004). In addition, it is herein pointed out that despite the advancedsequencing techniques available, ˜35-60% of the total protein-codinggenes display high similarities to “hypothetical proteins”, “predictedproteins” or “protein of unknown function” (Culligan, et al., 2014;Venter, et al., 2004).

3. Only the preselected clone is transformed into plants.

4. The expression and effect of a preselected clone in the target plantis unpredictable.

For the aforementioned reasons the novel method of the present inventionof screening plants transformed with an expression library for adesirable phenotype is advantageous.

It is herein acknowledged that drought and salinity are considered astwo abiotic stresses that have major effects on plant growth anddevelopment.

With respect to drought, it is considered the most devastatingenvironmental stress, which decreases crop growth and productivity.Drought severely affects plant growth and development with substantialreductions in growth rate and biomass accumulation. The mainconsequences of drought in plants are reduced rate of cell division andexpansion, leaf size, stem elongation and root proliferation, anddisturbed stomatal oscillations, and water use efficiency (WUE) (Farooqet al. 2009). This phenomenon involves genetic, physiological, andenvironmental events and their complex interactions. The rate and amountof plant growth depend on these events, which are affected by waterdeficit. Cell growth is one of the most drought-sensitive physiologicalprocesses due to the reduction in turgor pressure and water availability(Taiz and Zeiger, 2006). Under water deficiencies, cell elongation ofhigher plants can be inhibited by interruption of water flow from thexylem to the surrounding elongating cells. Impaired mitosis, reducedcell elongation and expansion result in reduced plant height, leaf areaand crop growth (Nonami, 1998).

Salinity is also considered one of the major severe abiotic factorsaffecting crop growth and productivity. During salt stress, all majorprocesses such as photosynthesis, protein synthesis and energy and lipidmetabolism are affected (Parida & Das, 2005). During initial exposure tosalinity, plants experience water stress, which in turn reduces leafexpansion. The osmotic effects of salinity stress can be observedimmediately after salt application and are believed to continue for theduration of exposure, resulting in inhibited cell expansion and celldivision, as well as stomatal closure. During long-term exposure tosalinity, plants experience ionic stress, which can lead to prematuresenescence of adult leaves, and thus a reduction in the photosyntheticarea available to support continued growth. In fact, excess sodium andmore importantly chloride has the potential to negatively affect plantenzymes, resulting in reduced energy production and other physiologicalchanges. It is further acknowledged that ionic stress results inpremature senescence of older leaves and in toxicity symptoms(chlorosis, necrosis) in mature leaves. Without wishing to be bound bytheory, the high sodium ions affect plants by disrupting proteinsynthesis and interfering with enzyme activity (Carillo et al., 2011).

The present invention provides a method for efficiently screening fornovel genes conferring resistance or improved tolerance to droughtand/or salinity in plants and especially in valuable crops.

The method of the present invention overcomes the above drawbacks byusing expressed genetic material (such as RNA or mRNA) that representthe genes that are being expressed in selected organisms, e.g. as aresult of environmental conditions (such as drought or high salt), andproducing a cDNA library that represents the ‘Meta-Expression’ ormetatranscriptome status of a certain biological niche or other geneticsource. The entire cDNA library is then transformed into plants andexpressed and screened for the desirable phenotype in the plants.

A core aspect of the present invention is that an expression library isproduced from various sources (including plants) and environments. Theexpression library is transformed into plants, which is the targetorganism in order to improve its traits or functions. The plantexpression library is then screened for the desirable trait, such assalt or drought resistance or tolerance, improved biomass and yieldproduction, biotic stresses (diseases and pathogens) resistance ortolerance, improved nutritional value or improved fertilizersutilization.

It is herein acknowledged that the environments (such as soils) in whichplants grow are inhabited by microbial communities, e.g. one gram ofsoil contains about 10⁷-10⁹ microbial cells (estimates of the number ofspecies of bacteria per gram of soil vary between 2000 and 8.3 million,https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2970868/) which compriseabout one gigabase of sequence information, or more. The microbialcommunities which inhabit environments in which plants grow (such assoils) are complex and remain poorly understood despite their economicimportance. Such microbial consortia provide the ecosystem necessary forplant growth, including fixing atmospheric nitrogen, nutrient cycling,disease suppression, and sequester iron and other metals.

It is within the scope of the present invention to use functionalmetagenomics and metatranscriptomics approaches to explore new geneswhich confer improved traits to plants.

Reference is now made to metagenomics approaches, employed by thepresent invention according to some aspects. Metagenomics is the studyof genetic material derived from environmental samples. It generallyrefers to as environmental genomics, eco-genomics or community genomics.While traditional microbiology and microbial genome sequencing andgenomics rely upon cultivated clonal cultures, environmental genesequencing cloned specific genes to produce a profile of diversity in anatural sample. In some aspects, metagenomics uses the study of thegenomes in a microbial community to constitute the first step tostudying the microbiome. Its main purpose is to infer the taxonomicprofile of a microbial community. The whole-metagenome sequencing (WMS)provides data on the functional profile of a microbial community. Suchwork revealed that the vast majority of microbial biodiversity had beenmissed by cultivation-based methods. In fact it is estimated that over99% of all microorganisms in almost every environment on earth cannot becultivated in the laboratory.

Metagenomics is herein also refers to metatranscriptomics, which studiesand correlates the transcriptomes of a group of interacting organisms orspecies. Metatranscriptomics involves sequencing the complete(meta)transcriptome of the microbial community. In some aspects,metatranscriptomics informs the genes that are expressed by thecommunity as a whole. With the use of functional annotations ofexpressed genes, it is possible to infer the functional profile of acommunity under specific conditions, which are usually dependent on thestatus of the host. While metagenomics provides data on the compositionof a microbial community under different conditions, metatrascriptomicsprovides data on the genes that are collectively expressed underdifferent conditions. Metatranscriptomics involves profiling ofcommunity-wide gene expression (RNA-seq). In specific aspects,metatranscriptomics describes the genes that are expressed in a specificmicrobial environment. Thus, metatranscriptomics is the study of thefunction and activity of the complete set of transcripts (RNA-seq) fromenvironmental samples.

It is noted that gene expression is log-like distributed, for example,top 100 genes of highest expression can cover up to 30% of alltranscripts. Even a single gene can cover up to 10%. Thus, a very highsequencing depth is required to see also lower expressed genes.

By using methods such as “shotgun” or PCR directed sequencing, largelyunbiased samples of the genes from the members of sampled communitiescan be obtained. It is herein acknowledged that metagenomics approachesprovide a powerful tool for utilizing microbial ecology to improvetraits in plants, for example, biological mechanisms that can beharnessed for agriculture and improved plant traits.

As used herein, the term “about” denotes ±25% of the defined amount ormeasure or value.

As used herein the term “similar” denotes a correspondence orresemblance range of about ±20%, particularly ±15%, more particularlyabout ±10% and even more particularly about ±5% .

As used herein the term “average” refers to the mean value as obtainedby measuring a predetermined parameter in each plant of a certain plantpopulation and calculating the mean value according to the number ofplants in said population.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a plant”includes one or more plants, reference to “a trait” includes one or moretraits and reference to “a cell” includes mixtures of cells, tissues,and the like.

A “plant” as used herein refers to any plant at any stage ofdevelopment, including a plant seed.

The term “plant” includes the whole plant or any parts or derivativesthereof, such as plant cells, plant protoplasts, plant cell tissueculture from which plants can be regenerated, plant callus or calli,meristematic cells, microspores, embryos, immature embryos, pollen,ovules, anthers, fruit, flowers, leaves, cotyledons, pistil, seeds, seedcoat, roots, root tips and the like.

The term “plant cell” used herein refers to a structural andphysiological unit of a plant, comprising a protoplast and a cell wall.The plant cell may be in a form of an isolated single cell or a culturedcell, or as a part of higher organized unit such as, for example, planttissue, a plant organ, or a whole plant.

The term “plant cell culture” or “tissue culture” as used herein meanscultures of plant units such as, for example, protoplasts, regenerablecells, cell culture, cells, cells in plant tissues, pollen, pollentubes, ovules, embryo sacs, zygotes and embryos at various stages ofdevelopment, leaves, roots, root tips, anthers, meristematic cells,microspores, flowers, cotyledons, pistil, fruit, seeds, seed coat or anycombination thereof.

The term “plant material” or “plant part” used herein refers to leaves,stems, roots, root tips, flowers or flower parts, fruits, pollen, eggcells, zygotes, seeds, seed coat, cuttings, cell or tissue cultures, orany other part or product of a plant or any combination thereof.

A “plant organ” as used herein means a distinct and visibly structuredand differentiated part of a plant such as a root, stem, leaf, flower,flower bud, or embryo.

“Plant tissue” as used herein means a group of plant cells organizedinto a structural and functional unit. Any tissue of a plant in plantaor in culture is included. This term includes, but is not limited to,whole plants, plant organs, plant seeds, tissue culture, protoplasts,meristematic cells, calli and any group of plant cells organized intostructural and/or functional units. The use of this term in conjunctionwith, or in the absence of, any specific type of plant tissue as listedabove or otherwise embraced by this definition is not intended to beexclusive of any other type of plant tissue.

As used herein, the term “trait” refers to a characteristic orphenotype, particularly, to a plant improving characteristic orphenotype. A phenotypic trait may refer to the appearance or otherdetectable characteristic of an individual, resulting from theinteraction of its genome, proteome and/or metabolome with theenvironment. For example, in the context of the present invention aplant improving trait or a desirable plant improving trait relates toresistance or tolerance to at least one biotic stress, resistance ortolerance to at least one abiotic stress, improved yield or biomass,improved grain yield, improved fertilizer uptake and usage efficiencyand any combination thereof.

A trait may be inherited in a dominant or recessive manner, or in apartial or incomplete-dominant manner. A trait may be monogenic (i.e.determined by a single locus) or polygenic (i.e. determined by more thanone locus) or may also result from the interaction of one or more geneswith the environment. A dominant trait results in a complete phenotypicmanifestation at heterozygous or homozygous state; conventionally, arecessive trait manifests itself only when present at homozygous state.

The term “phenotype” is understood within the scope of the presentinvention to refer to a distinguishable characteristic(s) of agenetically controlled trait.

As used herein, the phrase “phenotypic trait” refers to the appearanceor other detectable characteristic of an individual, resulting from theinteraction of its genome, proteome and/or metabolome with theenvironment.

It is within the scope of the current invention that “stress” may bedefined as any external factor that has a negative influence on plantgrowth, function and/or reproduction

The term “abiotic stress” is herein generally defined as the negativeimpact of non-living factors on the plant in a specific environment. Thenon-living variable must influence the environment beyond its normalrange of variation to adversely affect the plant or plant populationperformance or physiology in a significant way. Non limiting examples ofabiotic stress factors, or stressors, or environmental factors mayencompass factors such as sunlight, wind, temperature (cold, heat),salinity, over watering (flooding), drought and factors such asfertilizer uptake and fertilizer usage efficiency and any combinationthereof. Abiotic stress resistance or tolerance may enhance the growthand productivity of plants and specifically crops. It has been shownthat abiotic stressors are most harmful and may result in synergisticeffects when they occur together, in combinations of abiotic stressfactors.

The term “drought” refers hereinafter to a physical phenomenon generallycaused by an extended period of below average precipitation orirrigation. For example, not enough or low moisture (at the soil or atthe air), water supply shortages, dry soil, moisture regimes, highsalinity, heat and any combination thereof. Dry conditions may developfor different reasons. It can have a substantial impact on the ecosystemand agriculture, e.g. reduction in yield and crop damage.

Many organisms have drought tolerance physiological and geneticadaptations.

“Biotic stress” is herein defined as stress that occurs as a result ofdamage done to plants by other living organisms, such as bacteria,viruses, fungi, whitefly, thrips, spidermites, nematodes, parasites,beneficial and harmful insects, weeds, and cultivated or native plants.The types of biotic stresses imposed on a plant may be depended on bothgeography and climate and on the host plant and its ability to resistparticular stresses.

As used herein, the phrase “resistance” refers to the ability of a plantto restrict the growth and development of a specified pathogen and/orthe damage caused to the plant when compared to susceptible plants undersimilar environmental conditions. Resistant plants may exhibit somedisease symptoms or damage under pathogen or pest pressure or underabiotic stress condition.

It is further within the scope of the present invention that resistancemeans that a plant completely immunizes itself from a particular stress,for example to a biotrophic pathogen infection. According to specificembodiments of the invention, by transformation of an expression libraryto a host plant, the transformed host acquires a resistance gene whichprevents the proliferation of the pathogen and/or confers resistance toa particular abiotic stress (e.g. drought).

According to some aspects, resistance is an absolute term where theplant completely immunizes itself to a particular stress. It should benoted that this does not mean that tolerance cannot be obtained in caseof biotic or abiotic stress.

The term “tolerance” refers hereinafter to the characteristic of a plantthat allows a plant to avoid, tolerate or recover from biotic or abioticstressors, under conditions that would typically cause a greater amountof injury to other plants of the same species. These inheritablecharacteristics influence the degree of damage caused to the plant. Interms of agricultural production tolerance means that the plant can beunder stress (diseased/infected/or physiologically challenged) but theextent of loss does not exceed the economic threshold level (an extentof loss which do not hamper the economic potential of the produce).According to further aspects of the present invention, tolerance is arelative term. Examples of tolerance can be found in case of plantpathogens and all abiotic stresses, especially in the case of complextraits that are governed by multiple factors.

In general, ‘resistance’ and ‘tolerance’ are the terms used to denotethe ability of the plant to manage the stress, be it biotic or abiotic.

The term “transformation” used herein refers to genetic alteration ormodification induced by the introduction of exogenous DNA into a cell.This includes both integration of the exogenous DNA into the hostgenome, and/or introduction of plasmid DNA containing the exogenous DNAinto the plant cell. Such a transformation process results in theuptake, incorporation and expression of exogenous genetic material(exogenous DNA, for examples expression library prepared from ecologicalniche sampling). Plant transformation may refer to the introduction ofexogenous genes into plant cells, tissues or organs, employing direct orindirect means developed by molecular and cellular biology.

The term “environmental niche” or “ecological niche” generally refers tothe behavior of a species living under specific environmentalconditions. It includes the microbes, fungi, plants or other organismsthat inhabit a given environmental location (extremophiles). Theecological niche describes how an organism or population responds to thedistribution of resources and competitors and how it in turn altersthose same factors. The type and number of variables comprising thedimensions of an environmental niche vary from one species to anotherand the relative importance of particular environmental variables for aspecies may vary according to the geographic abiotic and bioticcontexts.

According to other aspects, the term “environmental niche” or“ecological niche” describes the relational position of a species orpopulation in an ecosystem. More specifically, it describes how apopulation responds to the abundance of its resources and competitorsand how it affects those same factors. The abiotic or physicalenvironment is also part of the niche because it influences howpopulations affect, and are affected by, resources and competition. Thedescription of a niche may include descriptions of the organism's lifehistory, habitat, and place in the food chain. In context of the presentinvention “environmental niche” or “ecological niche” can be definedaccording to biotic factors or abiotic factors such as high salinity,drought conditions, elevated heat, cold conditions, pH or any otherextreme environmental conditions.

It is within the scope of the current invention that the geneticmaterial is derived from a sampling of a predefined environmental niche,including from soil, water, plant biomass, microorganisms, yeast, algae,nematode, etc.

The term “microbiome” or “microbiota” as used herein refers to anecological community of commensal, symbiotic and pathogenicmicroorganisms found in and on all multicellular organisms from plantsto animals. A microbiota includes bacteria, archaea, protists, fungi andviruses. Microbiota has been found to be crucial for immunologic,hormonal and metabolic homeostasis of their host. The synonymous termmicrobiome describes either the collective genomes of the microorganismsthat reside in an environmental niche or the microorganisms themselves.The microbiome and host emerged during evolution as a synergistic unitfrom epigenetics and genomic characteristics, sometimes collectivelyreferred to as a holobiont.

The term “genetic material” or “genetic pool” refers hereinafter to sumof a population's genetic material at a given time. It includes allgenes and combinations of genes (sum of the alleles) in the population.

The term “isolated” as used hereinafter means that material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide which is separated from some or all of the coexistingmaterials in the natural system is isolated.

The nucleic acid isolated or derived from microorganisms or any organismcan preferably be inserted into a vector or a plasmid. Such vectors orplasmids are preferably those containing expression regulatorysequences, including promoters, enhancers and the like suitable forexpression in plants. Particularly preferred plasmids and methods forintroduction and transformation into them are described in detail in theprotocol set forth herein.

The term “expression library” as used hereinafter refers to a collectionof vectors or viruses (such as plant viruses used as virus-vectors) orplasmids or phages containing a representative sample of cDNA or genomicfragments that are constructed in such a way that they will betranscribed and or translated by the host organism (in the context ofthe present invention, plants). The technique uses expression vectors togenerate a library of clones, with each clone transcribing one RNA andor expressing one protein. This expression library is then screened forthe property of interest and clones of interest recovered for furtheranalysis. One and non-limiting example would be using an expressionlibrary to isolate genes that could confer resistance or tolerance todrought.

It is within the scope of the present invention that the expressionlibrary (usually derived from microbial genetic material) can beconstructed in a binary vector (or transfer DNA (T-DNA) binary system ora shuttle vector) able to replicate in multiple hosts (e.g. E. coli andAgrobacterium tumefaciens) to produce genetically modified plants. Theseare artificial vectors that have been created from the naturallyoccurring Ti plasmid found in Agrobacterium tumefaciens. In someaspects, the expression libraries are transferred from Agrobacteriumtumefaciens to plants.

The term “editing” or “gene editing” or “genome editing” refershereinafter to any conventional or known genome editing system or methodincluding systems using engineered nucleases selected from the groupconsisting of: meganucleases, zinc finger nucleases (ZFNs),transcription activator-like effector-based nucleases (TALEN), clusteredregularly interspaced short palindromic repeats (CRISPR) system and anycombination thereof. In the context of the present invention, theaforementioned gene editing techniques are used to edit a target gene ina desirable crop according to the information obtained from thetransgene identified by the method of the present invention.

The term “corresponding to the sequence” refers hereinafter to sequencehomology or sequence similarity. These terms relate to two or morenucleic acid or protein sequences, that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the available sequence comparison algorithms or by visual inspection.

According to further aspects of the invention, the term “correspondingto the nucleotide sequence” refers to variants, homologues and fragmentsof the indicated nucleotide sequence which possess or perform the samebiological function or correlates with the same phenotypiccharacteristic of the indicated nucleotide sequence.

Another indication that two nucleic acid sequences are substantiallysimilar or that a sequence is “corresponding to the nucleotide sequence”is that the two molecules hybridize to each other under stringentconditions. High stringency conditions, such as high hybridizationtemperature and low salt in hybridization buffers, permits onlyhybridization between nucleic acid sequences that are highly similar,whereas low stringency conditions, such as lower temperature and highsalt, allows hybridization when the sequences are less similar.

The term “similarity” or “sequence similarity” refers hereinafter to thedegree of resemblance between two sequences when they are compared. Thisis dependent on their identity and it shows the extent to which residuesare aligned. Sequence similarity refers to an optimal matching problem(i.e. for sequence alignments). The optimal matching algorithm finds theminimal number of edit operations (inserts, deletes, and substitutions)in order to align one sequence to another sequence. Sequence similaritysearches can identify “homologous” proteins or genes by detecting excesssimilarity, meaning, statistically significant similarity that reflectscommon ancestry.

It is within the scope of the current invention that similaritysearching is an effective and reliable strategy or tool for identifyinghomologs (i.e. sequences that share a common evolutionary ancestor). Nonlimiting examples of similarity searching programs, include BLAST (e.g.Altschul et al. 1997); units 3.3 and 3.4), PSI-BLAST (e.g. Altschul etal., 1997), SSEARCH (e.g. Smith and Waterman, 1981); Pearson, 1991, unit3.10), FASTA (e.g. Pearson and Lipman, 1988, unit 3.9) and the HMMER3(e.g. Johnson et al., 2010). Such programs produce accurate statisticalestimates, and can ensure that protein or nucleic acid sequences thatshare significant similarity also may have similar structures.Similarity searching is effective and reliable because sequences thatshare significant similarity can be inferred to be homologous; namelysharing a common ancestor.

Similarity is understood within the scope of the present invention torefer to a sequence similarity of at least 60%, particularly asimilarity of at least 70%, preferably more than 80% and still morepreferably more than 90%. The term “substantially similar” refers to anucleic acid, which is at least 50% identical in sequence to thereference when the entire ORF (open reading frame) is compared, wherethe sequence similarity is preferably at least 70%, more preferably atleast 80%, still more preferably at least 85%, especially more thanabout 90%, most preferably 95% or greater, particularly 98% or greater.

In some embodiments of the invention, such substantially similarsequences refer to polynucleotide or amino acid sequences that share atleast about 60% similarity, preferably at least about 80% similarity,alternatively, about 90%, 95%, 96%, 97%, 98% or 99% similarity to theindicated polynucleotide or amino acid sequence/s.

The present invention encompasses nucleotide sequences having at least60% similarity, preferably 70%, more preferably 80%, even morepreferable 90% and especially more preferable 95% similarity topolynucleotide sequences identified by the method of the presentinvention or to a reference sequence.

The present invention further encompasses amino acid sequences having atleast 60% similarity, preferably 70%, more preferably 80%, even morepreferable 90% and especially more preferable 95% similarity topolypeptide sequences identified by the method of the present inventionor to a reference sequence.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or gene orprotein sequence.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to the residues inthe two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins, it is recognizedthat residue positions which are not identical often differ byconservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule.

The term “identity” or “sequence identity” further refers hereinafter tothe amount of characters which match exactly between two differentsequences. Hereby, gaps are not counted and the measurement isrelational to the shorter of the two sequences.

In other words, if two sequences, which are to be compared with eachother, differ in length, sequence identity preferably relates to thepercentage of the nucleotide residues of the shorter sequence, which areidentical with the nucleotide residues of the longer sequence. As usedherein, the percent of identity between two sequences is a function ofthe number of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which needs tobe introduced for optimal alignment of the two sequences. The comparisonof sequences and determination of identity percent between two sequencescan be accomplished using a mathematical algorithm as known in therelevant art.

It is further within the scope that the terms “similarity” and“identity” additionally refer to local homology, identifying domainsthat are homologous or similar (in nucleotide and/or amino acidsequence). It is acknowledged that bioinformatics tools such as BLAST,SSEARCH, FASTA, and HMMER calculate local sequence alignments whichidentify the most similar region between two sequences. For domains thatare found in different sequence contexts in different proteins, thealignment should be limited to the homologous domain, since the domainhomology is providing the sequence similarity captured in the score.According to some aspects the term similarity or identity furtherincludes a sequence motif, which is a nucleotide or amino-acid sequencepattern that is widespread and has, or is conjectured to have, abiological significance. Proteins may have a sequence motif and/or astructural motif, a motif formed by the three-dimensional arrangement ofamino acids which may not be adjacent.

According to further embodiments, protein or polynucleotide sequenceswith specific location or domain sequence similarity are identified bythe method of the present invention. When comparing residues with noconservation the low similarity is meaningless thus lower overallsimilarity sequences with high conservation in conserved region will bestill considered as similar in a given range, for example of >60% (i.e.sequences showing low similarity of ˜37% to the nearest homolog butpossess all the conserved substrate binding residues of a specificprotein family) that can be found in hmm-based search algorithms such asHMMER3.

The term “Conserved Domain Database (CDD)” refers to a collection ofsequence alignments and profiles representing protein domains. It alsoincludes alignments of the domains to known 3-dimensional proteinstructures in the database (i.e. Molecular Modeling Database (MMDB).

In some embodiments of the invention, such substantially identicalsequences refer to polynucleotide or amino acid sequences that share atleast about 60% identity, preferably at least about 80% identity,alternatively, about 90%, 95%, 96%, 97%, 98% or 99% identity to theindicated polynucleotide or amino acid sequence/s.

Polypeptides within the scope of the present invention are at least 50%identical to the protein identified by the method of the presentinvention; or at least 55% identical, or at least 60% identical, or atleast 65% identical, or at least 70% identical, or at least 75%identical, or at least 80% identical, or at least 85% identical or atleast 90% identical or at least 95% identical to the protein identifiedby the method of the present invention or to a reference sequence.

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 80% sequenceidentity, preferably at least 85%, more preferably at least 90%, mostpreferably at least 95% sequence identity compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 80%, preferably atleast 85%, more preferably at least 90%, and most preferably at least95%. Preferably, optimal alignment is conducted using the homologyalignment algorithm of Needleman et al. (1970. J. Mol. Biol. 48:443).

The term “homolog” as used herein, refers to a DNA or amino acidsequence having a degree of sequence similarity in terms of shared aminoacid or nucleotide sequences. There may be partial similarity orcomplete similarity (i.e., identity). For protein sequences, amino acidsimilarity matrices may be used as are known in different bioinformaticsprograms (e.g. BLAST, FASTA, Bestfit program—Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive Madison, Wis. 53711, Smith Waterman).Different results may be obtained when performing a particular searchwith a different matrix. Degrees of similarity for nucleotide sequencesare based upon identity matches with penalties made for gaps orinsertions required to optimize the alignment, as is well known in theart (e.g. Altschul S. F. et al., 1990, J Mol Biol 215(3):403-10;Altschul S. F. et al., 1997, Nucleic Acids Res. 25:3389-3402). Guidancein determining which amino acid residues may be substituted, inserted,or deleted without abolishing biological or activity may be found usingcomputer programs well known in the art, for example, DNASTAR software.

The term “polymorphism” is understood within the scope of the inventionto refer to the presence in a population of two or more different formsof a gene, genetic marker, or inherited trait or a gene productobtainable, for example, through alternative splicing, DNA methylation,etc.

The present invention encompasses “High-throughput screening” or “HTS”technique, which herein refers to a method to rapidly identify genesthat modulate a particular biomolecular pathway or function. It includesmetatranscriptomic and metagenomic gene expression.

The present invention outlines a procedure for producing expressionlibraries from genetic material isolated from ecological niches, whichexpression libraries can be transformed into the target plant forscreening for a desirable trait such as tolerance or resistance tobiotic or abiotic stress and improving yield or biomass production.

According to one embodiment, the present invention provides a method forscreening for and identifying a desirable plant improving trait, themethod comprises steps of: (a) obtaining genetic material from asampling of a predefined environmental niche; and (b) constructing anexpression library from the genetic material. According to coreembodiments, the present invention further comprises steps of: (c)producing plants transformed with the expression library at anefficiency of at least 0.05% -30%, representing at least 10²-10¹⁰transgenes, thus creating the expressed library within the plants orseeds; (d) screening for transformed plants expressing the desirabletrait; and (e) identifying the transgene of the transformed plantsexpressing the desirable trait.

It is further within the scope to disclose the method as defined in anyof the above, wherein the step (a) further comprises steps of enrichingthe genetic material by growth on rich media or on selective media.

It is further within the scope to disclose the method as defined in anyof the above, wherein the step (a) further comprises steps of enhancingexpression of the desirable trait by culturing the genetic material onselective media for the desirable trait.

It is further within the scope to disclose the method as defined in anyof the above, wherein the step (b) comprises steps of producingprokaryotic cDNA library or eukaryotic cDNA library or both.

It is further within the scope to disclose the method as defined in anyof the above, wherein the step (b) further comprises steps of cloningthe cDNA library into at least one binary vector.

It is further within the scope to disclose the method as defined in anyof the above, wherein the binary vector comprises a constitutivepromoter or a stress induced promoter.

It is further within the scope to disclose the method as defined in anyof the above, wherein the binary vector comprises bacterial selectionmarker and plant transformation selection marker.

It is further within the scope to disclose the method as defined in anyof the above, wherein the bacterial selection marker is Kanamycinresistance, or any other antibiotic resistance conferring gene, and theplant transformation selection marker is bar gene, conferring resistanceto phosphinothricin containing herbicide (e.g. Basta herbicide).

Reference is now made to Glufosinate (also known as phosphinothricin andoften an ammonium salt) is a naturally occurring broad-spectrum systemicherbicide produced by several species of Streptomyces soil bacteria.Glufosinate is a broad-spectrum herbicide that is used to control weeds.It is sold in formulations under brands including Basta, Rely, Finale,Challenge and Liberty. The bar gene confers resistance to the herbicideBasta (containing phosphinothricin).

It is further within the scope to disclose the method as defined in anyof the above, further comprises steps of transforming the cloned binaryvectors into host cells.

It is further within the scope to disclose the method as defined in anyof the above, further comprises steps of transforming the cloned binaryvectors into Agrobacterium tumefaciens.

It is further within the scope to disclose the method as defined in anyof the above further comprises steps of introducing the transformedAgrobacterium tumefaciens into at least one of: whole plant, planttissue and plant cell.

It is further within the scope to disclose the method as defined in anyof the above, comprises steps of introducing the transformedAgrobacterium tumefaciens by spraying the plants with an inoculumcomprising transformed Agrobacterium.

It is further within the scope to disclose the method as defined in anyof the above, wherein the step (d) comprises growing the transformedplants under conditions selective for the desirable trait.

It is further within the scope to disclose the method as defined in anyof the above, further comprises steps of:

f. collecting T1 seeds from the transformed plants of step (d);g. determining seed library efficiency of the T1 seeds by calculatingratio of phosphinothricin resistant plants to total number of plants;h. sowing the T1 seeds of step (e) under selective conditions allowingscreening and selection of transformed plants expressing the desirabletrait;i. testing the selected plants expressing the desirable trait of step(g) for presence of the transgene; andj. isolating and sequencing the transgene of the selected transformedplants positively tested for the transgene of step (h).

It is further within the scope to disclose the method as defined in anyof the above, further comprises steps of

k. collecting T2 seeds from the plants of (h), which are found positivefor presence of the transgene;l. growing plants of the T2 seeds under selective conditions allowingscreening and selection of transformed plants expressing the desirabletrait as compared to control plants transformed with known genesconferring the desirable trait; andm. optionally, isolating and sequencing the transgene of the selectedplants of step (j).

It is further within the scope to disclose the method as defined in anyof the above, comprises steps of

-   -   a. recloning and sequencing the isolated transgene of step (i)        and/or (l);    -   b. transforming the recloned transgene into plants;    -   c. screening the transformed plants of step (b) for selection of        transformed plants expressing the desirable trait;    -   d. isolating the transgene from the selected plants of step (c);        and    -   e. optionally, repeating steps (a) to (d).

It is further within the scope to disclose the method as defined in anyof the above, wherein the environmental niche comprises samples derivedfrom ecological niches, sources, populations, habitats, gene pools,prokaryotic culture, eukaryotic culture and any combination thereof.

It is further within the scope to disclose the method as defined in anyof the above, wherein the environmental niche sampling comprisesmicrobiome, microbiota or microbial culture, plant, yeast, algae,nematode or any other organism or combinations thereof.

It is further within the scope to disclose the method as defined in anyof the above, wherein the environmental niche is defined according tobiotic factors, abiotic factors and a combination thereof.

It is further within the scope to disclose the method as defined in anyof the above, wherein the environmental niche sampling comprises soilsample, water sample, organic matter sample, any living organisms (suchas plant, yeast, bacteria, microorganism, algae, nematode) and anycombination thereof.

It is further within the scope to disclose the method as defined in anyof the above, wherein the desirable trait is selected from the groupconsisting of resistance or tolerance to at least one biotic stress,resistance or tolerance to at least one abiotic stress, improved yieldor biomass, improved grain yield, improved fertilizer uptake andimproved usage efficiency and a combination thereof.

It is further within the scope to disclose the method as defined in anyof the above, wherein the abiotic stress is selected from the groupconsisting of: drought, salinity, heat, cold, fertilizer uptake,fertilizer utilization efficiency and any combination thereof.

It is further within the scope to disclose the method as defined in anyof the above, wherein the biotic stress is selected from the groupconsisting of: pathogens, bacteria, viruses, fungi, parasites,beneficial and harmful insects, weeds, and cultivated or native plantsor any combination thereof.

It is further within the scope to disclose the method as defined in anyof the above, wherein the method comprises steps of extracting RNA fromthe sampling of the predefined environmental niche.

It is further within the scope to disclose the method as defined in anyof the above, wherein the RNA extraction is performed according tostandard commercial kits or according to any other protocol forextraction of RNA from environmental sampling.

It is further within the scope to disclose the method as defined in anyof the above, wherein the protocol for extraction of RNA fromenvironmental sampling comprises steps of:

-   -   a. obtaining a soil sample;    -   b. mixing the soil sample with an extraction buffer comprising        500 mM phosphate buffer pH 8 and 5% w/v cetyltrimethylammonium        bromide (CTAB) with phenol (pH 8)/chloroform/IAA ratio of        25:24:1;    -   c. subjecting the mixture of step (b) to 15 min shaking at        37° C. or to a bead beater for 1 min;    -   d. centrifuging the mixture of step (c) at 2,500 g for about 10        minutes at room temperature to obtain an aqueous phase;    -   e. transferring the aqueous phase into a new tube;    -   f. adding to the aqueous phase within the tube of step (e) an        equal amount of iso-propanol supplemented with 20 mg/ml crystal        violet solution to obtain violate stained solution;    -   g. mixing the solution by inverting said tube of step (f) and        then incubating the tube for about 30 minutes at room        temperature;    -   h. centrifuging the tube of step (g) at 2,500 g for about 30        minutes at room temperature to obtain a violet stained layer;    -   i. transferring the violate stained layer into a new tube and        centrifuging the tube for about 5 min at maximal speed to obtain        pellet and supernatant;    -   j. washing the pellet with 80% v/v ice cold ethanol and        centrifuging for additional 5 min to obtain pellet and        supernatant;    -   k. removing the supernatant of step (j) and allowing the pellet        to dry; and    -   l. suspending the dried pellet in water in a ratio of 100 μl        water to 2 gr of soil of step (a).

It is further within the scope to disclose polynucleotide sequencesobtainable by the method as defined above.

It is further within the scope to disclose the polynucleotide as definedabove, wherein the polynucleotide comprises a nucleotide sequencecorresponding to the sequence as set forth in a polynucleotide sequenceselected from the group consisting of SEQ ID NOs:1-148 and anycombination thereof.

It is further within the scope to disclose a polynucleotide sequencehaving at least 80%, 85%, 90% or 95% sequence similarity to apolynucleotide sequence obtainable by the method as defined above.

It is further within the scope to disclose a polypeptide sequenceobtainable by the method as defined above.

It is further within the scope to disclose the polypeptide sequence asdefined above, wherein the polypeptide comprises an amino acid sequencecorresponding to the sequence as set forth in a polypeptide sequenceselected from the group consisting of SEQ ID NOs: 149-321 and anycombination thereof.

It is further within the scope to disclose an amino acid sequence havingat least 60%, 70%, 80% or 90% sequence similarity to an amino acidsequence obtainable by the method as defined above.

It is further within the scope to disclose the use of the method asdefined above for identifying genes conferring resistance or toleranceto abiotic or biotic stress.

It is further within the scope to disclose the use of the method asdefined above for identifying genes conferring improved yield andbiomass, i.e. improved grain yield, in plants, for example by enhancinggrowth, with or without exposure to stress conditions.

It is further within the scope to disclose the use of the method asdefined above for identifying genes conferring improved yield.

It is further within the scope to disclose the use as defined in any ofthe above, wherein the abiotic stress is selected from the groupconsisting of: drought, salinity, heat, cold, fertilizer utilization,fertilizer uptake and any combination thereof.

It is further within the scope to disclose the use as defined in any ofthe above, wherein the biotic stress is selected from the groupconsisting of: pathogens, bacteria, viruses, fungi, parasites,beneficial and harmful insects, weeds, and cultivated or native plantsor any combination thereof.

It is further within the scope to disclose a method for screening forand identifying a drought resistance or tolerance improving trait inplants, the method comprises steps of: (a) obtaining genetic materialderived from a low moisture or a high salinity environmental nichesample; and (b) constructing expression library from the geneticmaterial. According to core embodiments, the method further comprisessteps of: (c) producing plants transformed with the expression libraryat an efficiency of at least 0.05% -30%, representing at least 10²-10¹⁰transgenes; (d) screening for transformed plants surviving predetermineddrought conditions; and (e) identifying the transgene of the droughtsurviving transformed plants of step (d).

It is further within the scope to disclose the method as defined above,wherein the step (b) further comprises steps of cloning the expressionlibrary into at least one binary vector.

It is further within the scope to disclose the method as defined in anyof the above, further comprises steps of:

-   -   f. collecting T1 seeds from the transformed plants of step (c);    -   g. sowing the T1 seeds in soil selective for transformed plants,        with water content of about 100% capacity;    -   h. growing plants of the T1 seeds in drought condition and/or        without irrigation until most of the plants die, to produce        transformed plants surviving the drought conditions;    -   i. growing the drought surviving transformed plants to produce        T2 seeds;    -   j. screening the drought surviving transformed plants of        step (i) for presence of a transgene;    -   k. isolating and sequencing the transgene from positively        screened plants of step (j);

It is further within the scope to disclose the method as defined in anyof the above, further comprises steps of

-   -   l. collecting T2 seeds from each of the transgene-containing        positively screened drought surviving transformed plants of step        (j);    -   m. growing T2 plants from each of the transgene-containing T2        seeds of step (l) under predetermined drought conditions as        compared to control plants transformed with known genes        conferring drought tolerance or drought resistance;    -   n. performing drought tolerance or resistance screen        measurements for each of the transgene-containing T2 plants as        compared to the control plants selected from the group        consisting of: turgor measurements, number of plants death,        state of plants and any combination thereof;    -   o. isolating the transgene from the screened drought resistance        performing T2 plants of step (n);    -   p. optionally, recloning the transgene into a binary vector;    -   q. optionally, transforming the cloned binary vector into plants        and growing the transformed plants under predetermined drought        conditions; and    -   r. optionally, repeating steps (l) to (q).

It is further within the scope to disclose the method as defined in anyof the above, wherein the step of growing T2 plants comprises steps of:(a) sowing the T2 seeds in soil selective for transformed plants, withwater content of about 100% capacity; and (b) irrigating the plants whenwater content in the soil reaches about 5-10%.

It is further within the scope to disclose the method as defined in anyof the above, wherein the predetermined drought conditions are selectedfrom the group consisting of low moisture, high salinity, dry soil andheat.

It is further within the scope to disclose polynucleotide sequencesobtainable by the method as defined in any of the above.

It is further within the scope to disclose the polynucleotide as definedabove, wherein the polynucleotide comprises a nucleotide sequencecorresponding to the sequence as set forth in a polynucleotide sequenceselected from the group consisting of SEQ ID NOs:1 to SEQ ID NO:148 andany combination thereof.

It is further within the scope to disclose polynucleotide sequenceshaving at least 80%, 85%, 90% or 95% sequence similarity topolynucleotide sequences obtainable by the method as defined in any ofthe above.

It is further within the scope to disclose a polypeptide sequenceobtainable by the method as defined in any of the above.

It is further within the scope to disclose the polypeptide sequence asdefined above, wherein the polypeptide sequence comprises an amino acidsequence corresponding to the sequence as set forth as set forth inpolypeptide sequence selected from the group consisting of SEQ. ID Nos:149-321 and any combination thereof

It is further within the scope to disclose polypeptide sequences havingat least 60%, 70%, 80% or 90% sequence similarity to amino acidsequences obtainable by the method as defined in any of the above.

It is further within the scope of the present invention to disclose amethod for extracting RNA from a soil sample comprising steps of:

-   -   m. obtaining a soil sample;    -   n. mixing said soil sample with an extraction buffer comprising        500 mM phosphate buffer pH 8 and 5% w/v cetyltrimethylammonium        bromide (CTAB) with phenol (pH 8)/chloroform/IAA ratios of        25:24:1;    -   o. subjecting said mixture of step (b) to about 15 min shake at        37° C. or to a bead beater for 1 min;    -   p. centrifuging said mixture of step (c) at 2,500 g for about 10        minutes at room temperature to obtain an aqueous phase;    -   q. transferring said aqueous phase into a new tube;    -   r. adding to said aqueous phase within said tube of step (e) an        equal amount of iso-propanol supplemented with 20 mg/ml crystal        violet solution to obtain violate stained solution;    -   s. mixing said solution by inverting said tube of step (f) and        then incubating said tube for about 30 minutes at room        temperature;    -   t. centrifuging said tube of step (g) at 2,500 g for about 30        minutes at room temperature to obtain a violet stained layer;    -   u. transferring said violate stained layer of step (h) into a        new tube and centrifuging said tube for about 5 min at maximal        speed to obtain pellet and supernatant;    -   v. washing said pellet with 80% v/v ice cold ethanol and        centrifuging for about additional 5 min to obtain pellet and        supernatant;    -   w. removing said supernatant of step (j) and the pellet is left        to dry; and    -   x. suspending said dried pellet in water in a ratio of 100 μl        water to 2 gr of soil of step (a).

It is further within the scope of the present invention to disclose amethod for screening for and identifying a desirable plant improvingtrait, said method comprises steps of:

-   -   y. obtaining a sampling of a predefined environmental niche;    -   z. extracting RNA from the sampling according to the method for        extracting RNA from a soil sample as defined above;    -   aa. constructing an expression library from the RNA of step (b);        The method further comprises steps of:    -   bb. producing plants transformed with the expression library at        an efficiency of at least 0.05% -30% representing at least        10²-10¹⁰ transgenes;    -   cc. screening for transformed plants expressing the desirable        trait; and    -   dd. identifying the transgene of the transformed plants        expressing the desirable trait.

It is further within the scope of the present invention to disclose anisolated polynucleotide having a nucleotide sequence corresponding tothe sequence as set forth in a polynucleotide sequence selected from thegroup consisting of SEQ ID NOs:1 to SEQ ID NO:148 and any combinationthereof.

It is further within the scope of the present invention to disclose anisolated polypeptide having an amino acid sequence corresponding to thesequence as set forth in polypeptide sequence selected from the groupconsisting of SEQ. ID Nos: 149-321 and any combination thereof.

In order to understand the invention and to see how it may beimplemented in practice, a plurality of preferred embodiments will nowbe described, by way of non-limiting example only, with reference to thefollowing examples.

EXAMPLE 1 A Process For Improving Traits in Plants by Transformation ofExpression Libraries From Ecological Niches Into Plants and ScreeningFor Desired Traits 1. Sample Collection and Processing

In the first step, genetic pools of a varied environmental samples andsources such as soil, water or organic matter from different habitatshave been isolated. The source is selected according to the specificdesired target traits. For example, when screening for drought orsalinity resistant gene, a dry land such as desert land or a highsalinity land or other enforcement will be used, but not necessarily.

The microbiome found in each sample may optionally be enriched by growthon rich media or selectively grown with antibiotics. To enhanceexpression of potentially desired genes, the culture is grown in stressconditions or media resembling, associated with or affecting the targettrait, such as salt or PEG rich media for drought or salinity resistancetrait.

Sample enrichment is carried on rich growth media (e.g. YPD) for severaldays at 28° C.-37° C. in shaker incubator. If eukaryotic libraries areprepared, anti-bacterial antibiotics such as Penicillin-Streptomycin andSpectinomycin are added.

To induce stress resistant genes, the sample is grown under any desiredenvironmental stress conditions. For example, to induce droughtresistance genes, the sample is grown under high osmotic stress byadding PEG to the growth media (10%-30% w/v). High salt concentrationmedia such as NaCl (5%-10% w/v) was used to induce high salinity stress.In addition, the samples are exposed to different nitrogen concentration(from 0-100 mM KNO₃ in water supplemented with 6 mM KH₂PO₄ and microelements, see Table 1,http://www.gatfertilizers.com/properties-of-solid-and-liquid-fertilizers/asrecommended by the manufacturer), extreme temperatures (50-60° C.) andany environmental stress desired.

TABLE 1 Element Percentage gr/Lt Iron 1.09 12.20 Manganese 0.48 5.47Zinc 0.15 1.75 Copper 0.05 0.55 Molybdenum 0.02 0.16 Boron 0.20 2.00

2. RNA Extraction

Total RNA extraction has been performed according to standard commercialkits such as RNeasy PowerSoil Total RNA Kit (Qiagen) and Quick-RNA (Zymoresearch). In addition, a unique protocol is used for extraction of RNAfrom soil samples, as follows:

In a 7 ml tube, 2 g of soil is disrupted with extraction buffer (500 mMPhosphate buffer pH 8 and 5% w/v CTAB with Phenol (pH 8), chloroform,IAA (25:24:1)). The tube is subjected to 15 min shaking at 37° C. or toa bead beater for 1 min. The tube is then centrifuged at 2,500 g for 10minutes at room temperature. The aqueous phase is transferred into a newtube and an equal amount of iso-propanol supplemented with 5 ul ofcrystal violate solution (20 mg/ml) is added. The tubes are mixed byinverting and left to stand for 30 minutes at room temperature, thencentrifuged at 2,500 g for 30 minutes at room temperature. The violatestained layer is transferred into a new 1.5 ml tube and centrifuged for5 min at maximal speed. The pellet is washed with 500 μl of 80%v/v icecold ethanol and centrifuged for additional 5 min. After centrifugation,the liquid is removed, and the pellet is left to dry. The dry pellet issuspended in 100 μl water.

3. Construction of cDNA Libraries

3.1. Eukaryotic cDNA Libraries

Eukaryotic cDNA libraries from total-RNA and mRNA are constructed basedon template switching-reverse transcription of poly-A mRNA (SMART) oroligo-capping rapid amplification of cDNA ends (5′-RACE) methods. Thereverse transcription of poly-A mRNA primers used are5′-ATTCTAGAGCGATCGCACATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3′ (referred toas SEQ. ID NO:321) and 5′-AAGCAGTGGTATCAACGCAGAGTGGCGCGCCrGrGG-3′(referred to as SEQ. ID NO:322). The oligo-capping rapid amplificationof cDNA ends primers used are5′-ATTCTAGAGCGATCGCACATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3′ (referred toas SEQ. ID NO:321) and 5′-InvddT (5′ Inverted Dideoxy-T)-r(AAGCAGUGGUAUCAACGCAGAGUGGCGCGCCG)-3′ (referred to as SEQ. ID NO: 323).The amplified cDNA is inserted into binary vectors (see FIGS. 1-4)between the promoter(s) (35S, KIN1, erd10 and/or CBF3) and the HSP orNOS terminator. FIG. 1A illustrates the pPA-35H vector, which has aconstitutive CaMV 35S promoter with the GFP gene cloned between thepromoter and terminator as an example. FIGS. 1B-D present vectorscontaining stress induced promoters from Arabidopsis thaliana: pPA-CHvector with CBF3 promoter having a nucleotide sequence as set forth inSEQ. ID NO: 330 (FIG. 1B), pPA-EH with Erd10 promoter having anucleotide sequence as set forth in SEQ. ID NO: 331 (FIG. 1C) and pPA-KHwith Kin1 promoter having a nucleotide sequence as set forth in SEQ. IDNO: 332 (FIG. 1D) with the GFP gene cloned between the promoter andterminator as an example (Plant Physiol. 1997 October; 115(2): 327-334.,Plant Journal (2004) 38, 982-993 incorporated herein by reference).

These vectors contain Kanamycin as a bacterial selection and the bargene as a transgenic plant selection conferring resistance to thephosphinothricin herbicide. At least one of the non-limiting examples ofGibson assembly, Restriction-ligation, Restriction free or In-Fusionmethods is used and then ligation products are transformed to E. colicompetent cells to grow under kanamycin selection. The library size isestimated by live count of transformed bacteria sown on LB petri dishes(usually 10{circumflex over ( )}5-10{circumflex over ( )}7) (FIG. 5).Vectors of the cDNA library are purified from E. coli bacteria withstandard mini-prep kits and transformed to electrocompetentAgrobacterium tumefaciens GV3101 cells. The transformed Agrobacteriumare grown on LB media under kanamycin and rifampicin selection (50 μg/mleach) over night at 28° C., (250 ml per 1 m² of target plant growtharea). The growth arrested on ice for at list 30 min and thencentrifuged for 10 min at 8000 rpm at 4° C. The pelleted Agrobacteriumare suspended in suspension buffer (5% sucrose and 0.03% L-77 Silwet,Momentive, US).

3.2. Prokaryotes cDNA Libraries

Prokaryotes cDNA libraries from total RNA are constructed based onstandard 5′ and 3′ RNA modifications with ScriptSeg™ Complete Kit(epicenter). Primers used are5′-ATTCTAGAGCGATCGCACATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3′ (referred toas SEQ. ID NO:321) and 5′-InvddT (5′ InvertedDideoxy-T)-r(AAGCAGUGGUAUCAACGCAGAGUGGCGCGCCG)-3′ (referred to as SEQ.ID NO:324). The amplified cDNA inserted into carrier vectors barringKanamycin and phosphinothricin resistance and then transformed to E.coli competent cells to grow under kanamycin selection (50 μg/ml). Thelibrary size is estimated by live count of transformed hosts (usually10{circumflex over ( )}5-10{circumflex over ( )}7). Vectors of the cDNAlibrary are purified from host cells with standard mini-prep kit (50 μl)and transformed to electrocompetent Agrobacterium GV3103 cells (100 μl).The transformed Agrobacterium are grown on LB media under kanamycin andrifampicin selection (50 μg/ml) over night at 28° C. (100 ml per 1 m² oftarget plant growth area). The growth is arrested on ice for at list 30min and then centrifuged for 5 min at 8000 rpm at 4° C. The pelletedAgrobacterium are suspended in suspension buffer (5% sucrose and 0.03%L-77 Silwet).

4. Growing and Transformation of Plants

4.1. Arabidopsis Plants

Plants are grown in controlled greenhouses as a preparation fortransformation. Plants are grown in soil composed of 75% peat, 25%perlite and are being irrigated routinely with water supplemented withfertilizer (e.g. Shefer 5.3.8, ICL Israel) according to manufacturerinstructions, as needed. Plants start flowering after 3-4 weeks and thenthey are ready for transformation. Transformed Agrobacterium withexpression libraries are grown as mentioned above and suspended insuspension buffer (5% sucrose and 0.03% L-77 Silwet) and are sprayed by2 liter sprayers (e.g. Solo, Germany) on the flowers. After 5-6 weeks ofcontinued growth when plants become dry, seeds are collected and kept ina cool dry place for 2 weeks or until used.

4.2. Tobacco Plants

Tobacco leaves are cut into 1-2 cm² pieces and sterilized by 70% ethanolfollowed by 0.3% bleach treatments for 5 minutes. Leaf pieces are mixedwith libraries transformed Agrobacterium (or with a any identified geneof SEQ ID 1-148 from Table 4), suspended in liquid Regeneration Medium(RM) supplemented with MS including Gamborg B5 vitamins, 3% sucrose, 2mg/L BAP (6-Benzylaminopurine) and 0.2 mg/L NAA (Naphthalene aceticacid) (e.g. Duchefa, Netherland) for 30 minutes. Bacteria are thanwashed and leaf pieces are placed on RM plant-agar plates for one day inthe dark. Leaf pieces are transferred to new selection RM plant-agarplates supplemented with 300 μg/ml of timentin antibiotic to kill theAgrobacterium and 1.5 μg/ml phosphoinotricin (e.g. Duchefa, Netherland)for selection of transgenic plants. FIG. 3A-B present a photographicillustration of tobacco tissue culture transformed with a library, 7days after transformation (FIG. 3A) and 40 days after transformation(FIG. 3B). After 6-8 weeks, plantlets start to appear and aretransferred to new vessels containing the same selection RM plant-agar,but BAP is excluded (see FIG. 3C). After rooting, plants are transferredto soil in the greenhouse.

EXAMPLE 2 A Process For Identifying Drought Resistance Traits in Plants

A. Screening for Drought and/or Salinity Resistant Plants/Genes

Arabidopsis T1 seeds harboring the desired expression library are beingused for the screen. At the first stage, the transformation efficiencyis defined for a specific seed library. 1 ml of seeds (˜50,000 seeds) isbeing sowed on soil irrigated with water supplemented with Basta (e.g.Bayer, Germany) according to manufacturer instructions. Seven days postsowing, the number of phosphinothricin resistant plants is counted andcompared with phosphinothricin susceptible plants (FIG. 4). Asdemonstrated in FIG. 4, the bigger plants are resistant tophosphinothricin while small plants are absent of the transgene andtherefore susceptible and will die. The seed library efficiency isrepresented by the ratio of the number of resistant plants to the numberof total plants.

The library is then sowed according to the desired number of plantsintended to be represented in the specific experiment and whichrepresents best the library size. For example, if an expression libraryconsists of 5×10⁴ genes, and the transformation efficiency is 1%, >5million seeds should be sowed. In this case, in ˜20 m² of soil, 50,000Basta resistant plants will be grown for the experiment.

Soil is irrigated once, when seeds are sown, with water supplementedwith phosphinothricin and fertilizer (e.g. Shefer 5.3.8, ICL Israel)according to manufacturer instructions, and soil water content reaches100% capacity. Plants are grown in air-conditioned controlledgreenhouses, and soil is not irrigated until most of the plants die fromlack of water. Surviving plants, ˜0.1% of initial phosphinothricinresistant plants, are being rescued by irrigation until they produceseeds which are being collected for T2 experiments. During their growth,the surviving plants are tested for their transgene, by gDNA extractionfrom one of their leaves and PCR using primers for the gene specificpromoters (CaMV 35S, CBF3, Erd10 and Kin1) and terminators (NOS, HSP)(see Table 2). PCR products are being sequenced and the resultedsequence is blasted versus sequence databases such as NCBI, both for DNAcomparisons (i.e. BLASTn) and for amino acid sequence comparisons (i.e.BLASTx).

Reference is now made to Table 2 presenting SEQ ID NOs of primer andpromoter sequences used in the present invention:

TABLE 2 SEQ ID NOs of primer sequences SEQ ID NO. Description SEQ ID NO:321 Reverse primer for transcription of poly-A mRNA SEQ ID NO: 322Forward primer for transcription of poly-A mRNA SEQ ID NO: 323 Forwardprimer for oligo-capping amplification of cDNA ends SEQ ID NO: 324Forward primer for amplification of prokaryote cDNA library (e.g.derived from total RNA) SEQ ID NO: 325 Forward primer for CaMV 35Spromoter SEQ ID NO: 326 Forward primer for CBF3 promoter SEQ ID NO: 327Forward primer for Erd10 promoter SEQ ID NO: 328 Forward primer for Kin1promoter SEQ ID NO: 329 Reverse primer for NOS/HSP terminator SEQ ID NO:330 CBF3 promoter SEQ ID NO: 331 Erd10 promoter SEQ ID NO: 332 Kin1promoterB. Subsequent Generations (T₂, T₃) Experiments

Seeds collected from drought surviving plants are being tested again infurther experiments including repeats and controls to test theirresistance/tolerance to drought (see FIG. 5).

Several genes were chosen to serve as controls in the droughtexperiments:

-   -   1) EGFP—jellyfish green fluorescent protein, cloned under the        control of the various used promoters and HSP terminator (see        vector maps of FIGS. 1A-D), is being served as a negative        control for drought, since it was not been shown to be        associated with improving plants resistance to drought (Yang        T-T, et al., 1996).    -   2) mtlD—mannitol-1-phosphate dehydrogenase from Escherichia        coli, cloned under the control of the various used promoters and        HSP terminator (see vector maps of FIGS. 1A-D), is being served        as a positive control since it was shown to be associated with        improving plants resistance to drought and salt (Hema R. et al.,        2014).    -   3) HRD—The HARDY gene from Arabidopsis thaliana cloned under the        control of the various used promoters and HSP terminator (see        vector maps of FIGS. 1A-D), is being served as a positive        control since was shown to be associated with improving plants        resistance to drought and salt (Karaba A, et al., 2007).

Plants identified as expressing unique genes in the screen experiments,including all controls, are sown in trays 38×28 cm with 16 plasticinserts in each tray (e.g. Desch Plantpak, Netherland), filled with soilsupplemented with fertilizer and phosphinothricin as above. In eachinsert several seeds are sown and after 10 days a singlephosphinothricin resistant plant is being kept for further experiments.Each experiment contains 20-40 repeats of each plant, representing theexpressed unique genes, which are spread in random on the greenhousetables. Irrigation of the soil is similar to the screen experiment; itis done when the seeds are sown, except when soil is completely dry andreaches weight lower then initial weight of soil before irrigation(˜5%-10% of water content), then plants are irrigated again to checkrevival performance.

Reference is now made to FIG. 6 showing photographic results ofscreening for transgenic plants resistance to drought grown under theconditions as described above. This figure shows that transgenic plantscarrying drought resistance genes 10, 20 and 30 survive in severedrought conditions, while other transgenic plants that do not harbordrought resistant conferring genes do not survive the stress conditions.It is noted that within the small area shown in this figure (˜15×25 cm),about 300 plants were screened while 3 survived the drought conditions.

When drought conditions start to develop, various measurements aretaken, as shown in Table 3:

1) turgor observation, measured by scale of 1-10, when 1 is high turgorand 10 is total loss of turgor (see FIG. 7).

2) Weight of plant and pot, by scale in grams.

3) Death of plants observation, 10=dead and 1=alive (see FIG. 8)

4) State of plants observation in a scale of 1-10, when 1 is good stateand 10 is poor.

TABLE 3 Measurements taken in drought experiments measurement units timeof measurement Weight of pot Grams Start till end of experiment TurgorObservation units 1-10, From beginning of turgor where 1 is 0 turgorloss and loss (~15-27 days from last 10 is 100% turgor loss irrigation)Death Observation units 1-10 From first death observed State of plantsObservation units 1-10 During first 2 weeks and one day after revival

Reference is now made to FIG. 7, showing loss of turgor pressure inplants expressing genes used as control relative to soil water content(dark gray), days after cessation of irrigation. This figure showscurves of Arabidopsis plants, expressing different genes (indicated), asa response to growth under drought conditions. Dark line indicates soilwater content from 40% in day 15 after water irrigation ceased, to closeto 0% at day 22 after water irrigation ceased. The negative control GFPplant's loss of turgor pressure response is similar to that of HRDexpressing plants, while mtlD expressing plants turgor pressure, seem tobe less effected by drought until day 20 after water irrigation ceased.

It is demonstrated in this figure that plants expressing the positivecontrol genes mtlD and HRD showed improved resistance to drought byshowing significantly reduced loss of turgor pressure effects, whiletransgenic plants expressing the negative control GFP gene showedelevated loss of turgor pressure effect when exposed to the same watercontent loss.

Reference is now made to FIG. 8 showing normalized death scale ofpositive control expressing transgenic plants as compared to GFPexpressing plants. As can be seen plants expressing the droughtresistance positive control genes HRD and mtlD showed significantlyreduced death rate as compared to the negative control GFP expressingplants.

Reference is now made to FIG. 9 graphically showing phenotypic resultsof several drought resistance genes identified by the method of thepresent invention.

The graph shows average results of turgor pressure (Tu) and death rate(Dr) for several identified genes (see Table 4) under severe droughtconditions. Scale for death and turgor loss is 1-10 when 10 isconsidered dry-brown and dead plants, or total loss of turgor,respectively. The results in the graph represent day 23 (1), day 28 (6)and day 30 (8) from sowing. Each column for each of the differentexpressed genes represents average of 5 repeats with 4 plants in eachrepeat. GFP expressing plants served as negative control and HRD aspositive control. As can be seen, all tested genes identified by themethod of the present invention showed significantly reduced turgor loss(by at least two fold after about 23 days from sowing) and reduced deathrate (in the range of 9 to 2 fold after 30 days from sowing) as comparedto plants expressing the negative control GFP gene. Moreover, plantsexpressing the newly discovered genes (see Table 4) demonstrated asignificantly reduced death rated as compared to the positive controlHRD expressing plants. These results indicate that by the method of thepresent invention, newly drought resistance genes are identified, whichconfer improved tolerance to drought in plants.

Another method used for evaluating plants performance in droughtconditions is measuring their leaf area during the growth phase whendrought conditions become prominent. About 10-14 days from sowing theplants, plant images were taken every 2-3 days together with a 50 mm²white surface. Image analysis was performed on pictures taken from thedrought experiments and leaf area was calculated. The leaf area ofseveral plant lines expressing novel genes identified as conferringdrought resistance after re-cloning was compared to positive andnegative controls (see Table 5 and FIG. 10).

The graph of FIG. 10 shows image analysis of leaf area of transformedplant lines. Two independent transformation events of the identifiedgene having SEQ ID NO:16 (FIG. 10A) and two independent transformationevents of the identified gene having SEQ ID NO:25 (FIG. 10B) are shownin darker lines on top of each of the FIGS. 10A and 10B. Thesetransgenic plants are compared to negative control plants expressingGFP, and positive control plants expressing mtlD, shown in lighter graylines on the botom of each of the FIGS. 10A and 10B. Improvedperformance under drought is shown as percentage from control plants atthe indicated measured timepoint (TP) (arrows and percentages shown inthe figure).

As can be seen in this figure, the total leaf area of plants expressingthe newly identified tested genes was increased by between about 10% andabout 82% (e.g. by about 45%) relative to plants expressing negativecontrol genes.

To conclude, the present invention provides newly identified genesdemonstrated to confer tolerance to drought conditions in plants.

C. Re-Cloning and Retransformation of Selected Genes Into Plants

Selected genes from section B are re-cloned into the binary vectors asdescribed above (i.e. FIG. 1A-D) and sequenced to confirm that it hasthe same sequence as the original gene from T1 and T2 experiments.Plants are transformed with the re-cloned gene and seeds are collected.Experiments are repeated as in B except for each gene 3-5 individualtransgenic plants with different unrelated transformation events aretested. Each individual transgenic plant/event is subjected to 5-10times of repeats in experiments, hence for each event for every gene20-40 plants are tested, and for every different gene 60-200 plants aretested.

EXAMPLE 3 Polynucleotide Sequences Identified as Improving Droughtand/or Salinity Resistance in Plants

The process described above of screening of T1 transgenic seeds revealedabout 1000 transgenes as candidate polynucleotide sequences forimproving drought resistance in plants. Of these candidates, thescreening of T2 seeds revealed about 140 best performing transgenespotentially improving drought resistance or tolerance in plants. Thesetransgene sequences are subjected to further validation tests.

Reference is now made to Table 4, presenting examples of novel andunique polynucleotide sequences and polypeptides encoded by thesesequences, found by the method of the present invention. These sequencesare metatranscriptomes purified from environmentally challenged niches,

SEQ ID NO:1 to SEQ ID NO:148 represent polynucleotide sequences found bythe method of the present invention as candidates for improving droughtresistance in plants (Table 4).

SEQ ID NO:149 to SEQ ID NO:321 represent polypeptide sequences encodedby the corresponding polynucleotide sequence found by the method of thepresent invention as candidates for improving drought resistance inplants (see Table 4).

Note that DNA sequences SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:43, SEQ ID NO:47, SEQ IDNO:51, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:60, SEQ ID NO:75, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:97, SEQ ID NO:98, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ IDNO:109, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:120, SEQ ID NO:132, SEQID NO:134, SEQ ID NO:135, SEQ ID NO:140, SEQ ID NO:141 encode more thanone open reading frame (ORF) (referred to as SEQ. ID NO X.1p and X.2petc.) depending on different start codons.

TABLE 4 SEQ ID Nos of polynucleotide and polypeptide sequencesPolynucleotide SEQ ID NO. Polynucleotide name SEQ ID NO: 1 A454 SEQ IDNO: 2 A456 SEQ ID NO: 3 A458.1 SEQ ID NO: 4 A458.2 SEQ ID NO: 5 A460 SEQID NO: 6 A462 SEQ ID NO: 7 A463 SEQ ID NO: 8 A466 SEQ ID NO: 9 A468 SEQID NO: 10 A470 SEQ ID NO: 11 A475 SEQ ID NO: 12 A477 SEQ ID NO: 13 A480SEQ ID NO: 14 A481 SEQ ID NO: 15 A483 SEQ ID NO: 16 A484 SEQ ID NO: 17A485a SEQ ID NO: 18 A485b SEQ ID NO: 19 A486 SEQ ID NO: 20 A498 SEQ IDNO: 21 A499 SEQ ID NO: 22 A501 SEQ ID NO: 23 A504.1 SEQ ID NO: 24 A504SEQ ID NO: 25 A506 SEQ ID NO: 26 A507.1 SEQ ID NO: 27 A507.2 SEQ ID NO:28 A510a SEQ ID NO: 29 A510b SEQ ID NO: 30 A512 SEQ ID NO: 31 A513a SEQID NO: 32 A513b SEQ ID NO: 33 A518 SEQ ID NO: 34 A520a SEQ ID NO: 35AC2510 SEQ ID NO: 36 AD2607.1 SEQ ID NO: 37 AD2607.3 SEQ ID NO: 38 D860aSEQ ID NO: 39 D860b SEQ ID NO: 40 D862 SEQ ID NO: 41 D863 SEQ ID NO: 42D881 SEQ ID NO: 43 D890 SEQ ID NO: 44 De203 SEQ ID NO: 45 De214a SEQ IDNO: 46 De215a SEQ ID NO: 47 De215b.1 SEQ ID NO: 48 De215b.4 SEQ ID NO:49 De215c SEQ ID NO: 50 De217 SEQ ID NO: 51 De223a SEQ ID NO: 52 De223bSEQ ID NO: 53 De227 SEQ ID NO: 54 De239a SEQ ID NO: 55 De245 SEQ ID NO:56 De250.1 SEQ ID NO: 57 De250.2 SEQ ID NO: 58 De251 SEQ ID NO: 59 De313SEQ ID NO: 60 F1022a SEQ ID NO: 61 F1022b SEQ ID NO: 62 G1085a SEQ IDNO: 63 G1181 SEQ ID NO: 64 G1190 SEQ ID NO: 65 H1301.1 SEQ ID NO: 66H1301.2 SEQ ID NO: 67 K1464 SEQ ID NO: 68 K1475 SEQ ID NO: 69 M603 SEQID NO: 70 M606.1 SEQ ID NO: 71 M606.2 SEQ ID NO: 72 M607.1 SEQ ID NO: 73M607.2 SEQ ID NO: 74 M609a.1 SEQ ID NO: 75 M609a.2 SEQ ID NO: 76 M609bSEQ ID NO: 77 M619a SEQ ID NO: 78 M619b SEQ ID NO: 79 M622a SEQ ID NO:80 M622b SEQ ID NO: 81 M623a SEQ ID NO: 82 M623b.1 SEQ ID NO: 83 M623b.3SEQ ID NO: 84 M623c SEQ ID NO: 85 M624 SEQ ID NO: 86 M625a.3 SEQ ID NO:87 M625a SEQ ID NO: 88 M625b SEQ ID NO: 89 M631 SEQ ID NO: 90 M632a SEQID NO: 91 M635.1 SEQ ID NO: 92 M635.2 SEQ ID NO: 93 M638 SEQ ID NO: 94M643 SEQ ID NO: 95 M649 SEQ ID NO: 96 M650a.3 SEQ ID NO: 97 M650a SEQ IDNO: 98 M650b SEQ ID NO: 99 M657 SEQ ID NO: 100 M659a SEQ ID NO: 101 M661SEQ ID NO: 102 M663 SEQ ID NO: 103 M664.1 SEQ ID NO: 104 M664.2 SEQ IDNO: 105 M666 SEQ ID NO: 106 M671 SEQ ID NO: 107 M673 SEQ ID NO: 108M676.3 SEQ ID NO: 109 M676 SEQ ID NO: 110 M677a SEQ ID NO: 111 M677b.1SEQ ID NO: 112 M677b.3 SEQ ID NO: 113 M680 SEQ ID NO: 114 M691a.1 SEQ IDNO: 115 M691a.2 SEQ ID NO: 116 M691b SEQ ID NO: 117 M693 SEQ ID NO: 118M697 SEQ ID NO: 119 M698 SEQ ID NO: 120 M705 SEQ ID NO: 121 M706 SEQ IDNO: 122 M715a SEQ ID NO: 123 M715b SEQ ID NO: 124 M719 SEQ ID NO: 125M724 SEQ ID NO: 126 N1503a SEQ ID NO: 127 N1527.1 SEQ ID NO: 128 N1527.2SEQ ID NO: 129 N1529 SEQ ID NO: 130 N1530 SEQ ID NO: 131 P1611 SEQ IDNO: 132 P1620.1 SEQ ID NO: 133 P1620.3 SEQ ID NO: 134 P1623a SEQ ID NO:135 P1623b SEQ ID NO: 136 P1625a SEQ ID NO: 137 P1625b SEQ ID NO: 138P1731 SEQ ID NO: 139 P1744 SEQ ID NO: 140 P1747.1 SEQ ID NO: 141 P1747.3SEQ ID NO: 142 SN8 SEQ ID NO: 143 V1906b SEQ ID NO: 144 V1906c SEQ IDNO: 145 V1907a SEQ ID NO: 146 V1907b SEQ ID NO: 147 X2005 SEQ ID NO: 148X2026 SEQ ID NO: 149 A454p SEQ ID NO: 150 A456p SEQ ID NO: 151 A458.1pSEQ ID NO: 152 A458.2p SEQ ID NO: 153 A460p SEQ ID NO: 154 A462p SEQ IDNO: 155 A463p SEQ ID NO: 156 A466p SEQ ID NO: 157 A468.1p SEQ ID NO: 158A468.2p SEQ ID NO: 159 A470p SEQ ID NO: 160 A475.1p SEQ ID NO: 161A475.2p SEQ ID NO: 162 A477p SEQ ID NO: 163 A480p SEQ ID NO: 164 A481pSEQ ID NO: 165 A483p SEQ ID NO: 166 A484p SEQ ID NO: 167 A485ap SEQ IDNO: 168 A485bp SEQ ID NO: 169 A486p SEQ ID NO: 170 A498.1p SEQ ID NO:171 A498.2p SEQ ID NO: 172 A499.1p SEQ ID NO: 173 A499.2p SEQ ID NO: 174A501p SEQ ID NO: 175 A504.1p SEQ ID NO: 176 A504.2p SEQ ID NO: 177 A506pNo ORF identified No ORF identified SEQ ID NO: 178 A507.2p SEQ ID NO:179 A510a.1p SEQ ID NO: 180 A510a.2p No ORF identified No ORF identifiedSEQ ID NO: 181 A512p SEQ ID NO: 182 A513ap SEQ ID NO: 183 A513bp SEQ IDNO: 184 A518p SEQ ID NO: 185 A520ap SEQ ID NO: 186 AC2510ap SEQ ID NO:187 AD2607.1p SEQ ID NO: 188 AD2607.2p SEQ ID NO: 189 AD2607.3p SEQ IDNO: 190 D860ap SEQ ID NO: 191 D860bp SEQ ID NO: 192 D862p SEQ ID NO: 193D863p SEQ ID NO: 194 D881p SEQ ID NO: 195 D890.1p SEQ ID NO: 196 D890.2pSEQ ID NO: 197 De203p SEQ ID NO: 198 De214ap SEQ ID NO: 199 De215ap SEQID NO: 200 De215b.1p SEQ ID NO: 201 De215b.2p SEQ ID NO: 202 De215b.3pSEQ ID NO: 203 De215b.4p SEQ ID NO: 204 De215cp No ORF identified No ORFidentified SEQ ID NO: 205 De223a.1p SEQ ID NO: 206 De223a.2p SEQ ID NO:207 De223bp SEQ ID NO: 208 De227p SEQ ID NO: 209 De239a.1p SEQ ID NO:210 De239a.2p SEQ ID NO: 211 De245.1p SEQ ID NO: 212 De245.2p SEQ ID NO:213 De250p SEQ ID NO: 214 De250.2p SEQ ID NO: 215 De251p SEQ ID NO: 216De313p SEQ ID NO: 217 F1022a.1p SEQ ID NO: 218 F1022a.2p SEQ ID NO: 219F1022bp SEQ ID NO: 220 G1085ap SEQ ID NO: 221 G1181p SEQ ID NO: 222G1190p SEQ ID NO: 223 H1301.1p SEQ ID NO: 224 H1301.2p No ORF identifiedNo ORF identified SEQ ID NO: 225 K1475p SEQ ID NO: 226 M603p SEQ ID NO:227 M606.1p SEQ ID NO: 228 M606.2p SEQ ID NO: 229 M607. 1p SEQ ID NO:230 M607.2p SEQ ID NO: 231 M609a.1p SEQ ID NO: 233 M609a.3p SEQ ID NO:232 M609a.2p SEQ ID NO: 234 M609bp SEQ ID NO: 235 M619ap SEQ ID NO: 236M619b.1p SEQ ID NO: 237 M619b.2p SEQ ID NO: 238 M622ap SEQ ID NO: 239M622b.1p SEQ ID NO: 240 M622b.2p SEQ ID NO: 241 M623a.1p SEQ ID NO: 242M623a.2p No ORF identified No ORF identified SEQ ID NO: 243 M623b.3p SEQID NO: 244 M623cp SEQ ID NO: 245 M624. 1p SEQ ID NO: 246 M624.2p SEQ IDNO: 249 M625a.3p SEQ ID NO: 247 M625a.1p SEQ ID NO: 248 M625a.2p SEQ IDNO: 250 M625bp SEQ ID NO: 251 M631p SEQ ID NO: 252 M632ap SEQ ID NO: 253M635.1p SEQ ID NO: 254 M635.2p SEQ ID NO: 255 M638p SEQ ID NO: 256 M643pSEQ ID NO: 257 M649p SEQ ID NO: 260 M650a.3p SEQ ID NO: 258 M650a.1p SEQID NO: 259 M650a.2p SEQ ID NO: 261 M650b.1p SEQ ID NO: 262 M650b.2p SEQID NO: 263 M657p SEQ ID NO: 264 M659ap SEQ ID NO: 265 M661p SEQ ID NO:266 M663p SEQ ID NO: 267 M664.1p SEQ ID NO: 268 M664.2p SEQ ID NO: 269M666.1p SEQ ID NO: 270 M666.2p SEQ ID NO: 271 M671.1p SEQ ID NO: 272M671.2p SEQ ID NO: 273 M673.1p SEQ ID NO: 274 M673.2p SEQ ID NO: 277M676.3p SEQ ID NO: 275 M676.1p SEQ ID NO: 276 M676.2p SEQ ID NO: 278M677ap SEQ ID NO: 279 M677b.1p SEQ ID NO: 280 M677b.2p SEQ ID NO: 281M677b.3p SEQ ID NO: 282 M677b.4p SEQ ID NO: 283 M680p SEQ ID NO: 284M691a.1p SEQ ID NO: 285 M691a.2p SEQ ID NO: 286 M691bp SEQ ID NO: 287M693p SEQ ID NO: 288 M697p SEQ ID NO: 289 M698p SEQ ID NO: 290 M705.1pSEQ ID NO: 291 M705.2p SEQ ID NO: 292 M706p SEQ ID NO: 293 M715ap SEQ IDNO: 294 M715bp SEQ ID NO: 295 M719p SEQ ID NO: 296 M724p SEQ ID NO: 297N1503ap SEQ ID NO: 298 N1527.1p SEQ ID NO: 299 N1527.2p SEQ ID NO: 300N1529p SEQ ID NO: 301 N1530p SEQ ID NO: 302 P1611p SEQ ID NO: 303P1620.1p SEQ ID NO: 304 P1620.2p SEQ ID NO: 305 P1620.3p SEQ ID NO: 306P1623a.1p SEQ ID NO: 307 P1623a.2p SEQ ID NO: 308 P1623b.1p SEQ ID NO:309 P1623b.2p SEQ ID NO: 310 P1625ap SEQ ID NO: 311 P1625bp SEQ ID NO:312 P1731p SEQ ID NO: 313 P1744p SEQ ID NO: 314 P1747.1p SEQ ID NO: 315P1747.2p SEQ ID NO: 316 P1747.3p SEQ ID NO: 317 P1747.4p No ORFidentified No ORF identified SEQ ID NO: 318 V1906bp No ORF identified NoORF identified SEQ ID NO: 319 V1907ap No ORF identified No ORFidentified SEQ ID NO: 320 X2005p SEQ ID NO: 321 X2026p

Reference is now made to Table 5 presenting phenotypic results ofseveral of the identified genes in the drought tolerance experiments.Plants were grown in soil in controlled greenhouses and tested fordrought tolerance under the conditions mentioned above. During theirgrowth, measurements and images were taken (see Table 3) and imageanalysis was applied converting the images to leaf area per plant.Results are shown as percentage of GFP expressing plants measurementsthat served as a negative control during the drought phase.

TABLE 5 Results of drought experiments conducted with T2 Arabidopsisplants Seq ID DR ±SD Seq ID DR ±SD Seq ID DR ±SD Seq ID DR ±SD Seq ID DR±SD SEQ ID 116.00  3.13 SEQ ID 137.14  7.05 SEQ ID 144.00  5.56 SEQ ID143.23  7.26 SEQ ID 120.00 10.25 NO: 1 NO: 25 NO: 55 NO: 91/92 NO: 122SEQ ID 132.86  6.68 SEQ ID 135.00  7.64 SEQ ID 134.00  3.28 SEQ ID133.33  6.84 SEQ ID 130.73  5.93 NO: 2 NO: 26/27 NO: NO: 93 NO: 12456/57 SEQ ID 151.43 10.52 SEQ ID 187.64 11.00 SEQ ID 151.6 20.7 SEQ ID102.50  7.08 SEQ ID 139.10  9.76 NO: 17/18 NO: 28 NO: 58 NO: 94 NO: 125SEQ ID 146.9 20.6 SEQ ID 118.57  1.88 SEQ ID 134.08  4.45 SEQ ID 133.41 7.61 SEQ ID 119.09  4.03 NO: 5 NO: 30 NO: 60 NO: 97 NO: 127/ 128 SEQ ID118.57  5.26 SEQ ID 112.56  4.97 SEQ ID  99.72  4.71 SEQ ID 137.50  8.80SEQ ID 135.01  7.89 NO: 6 NO: 31 NO: 62 NO: 99 NO: 129 SEQ ID 156.7 23.4SEQ ID 167.57  7.20 SEQ ID 277.71 16.80 SEQ ID 160.11 20.12 SEQ ID196.80  9.06 NO: 7 NO: 33 NO: 63 NO: 100 NO: 130 SEQ ID 162.1 17.1 SEQID 118.92  5.31 SEQ ID 136.83  6.62 SEQ ID 182.50 10.00 SEQ ID 113.98 7.65 NO: 8 NO: 34 NO: 64 NO: 101 NO: 131 SEQ ID 138.24 20.36 SEQ ID115.20  6.60 SEQ ID 107.77 10.82 SEQ ID 136.67  8.72 SEQ ID 110.33  6.64NO: 9 NO: 35 NO: NO: 102 NO: 132/ 65/66 133 SEQ ID 116.00  3.19 SEQ ID109.71  7.79 SEQ ID 131.25  7.04 SEQ ID 121.79  7.45 SEQ ID 107.54  9.76NO: 10 NO: 36/37 NO: 67 NO: NO: 134 103/104 SEQ ID 107.14  2.93 SEQ ID124.59  6.74 SEQ ID 186.67  9.85 SEQ ID 126.73  6.48 SEQ ID 114.17  5.84NO: 11 NO: 38 NO: 68 NO: 105 NO: 137 SEQ ID 122.86  4.53 SEQ ID 154.2910.83 SEQ ID 132.64  8.96 SEQ ID 125.00  5.94 SEQ ID 139.80  9.87 NO: 12NO: 40 NO: 69 NO: 106 NO: 138 SEQ ID 160.00 12.32 SEQ ID 117.14  5.71SEQ ID 145.00  7.07 SEQ ID 130.00  6.64 SEQ ID 115.04  6.38 NO: 13 NO:41 NO: NO: 107 NO: 139 70/71 SEQ ID 142.86  8.37 SEQ ID 118.27  3.56 SEQID 134.08  7.08 SEQ ID 175.00  7.38 SEQ ID 105.73  8.08 NO: 14 NO: 42NO: NO: 109 NO: 140/ 72/73 141 SEQ ID 145.71  7.24 SEQ ID 141.69  8.03SEQ ID 187.50 10.00 SEQ ID 118.92  5.89 SEQ ID 141.43  3.65 NO: 15 NO:43 NO: NO: 110 NO: 142 74/75/76 SEQ ID 136.13  8.55 SEQ ID 144.00  6.36SEQ ID 125.00  8.29 SEQ ID 113.14  8.47 SEQ ID 115.50  7.96 NO: 16 NO:44 NO: 77 NO: 113 NO: 143 SEQ ID 108.33  2.73 SEQ ID 142.70  9.33 SEQ ID123.73  6.78 SEQ ID 108.04  5.44 SEQ ID 112.59  7.32 NO: 17 NO: 45 NO:79 NO: 114/ NO: 145 115/116 SEQ ID 121.67  5.66 SEQ ID 119.36  9.40 SEQID 159.79  8.45 SEQ ID 167.50  9.13 SEQ ID 121.66  8.81 NO: 19 NO: 46NO: 81 NO: 117 NO: 147 SEQ ID 118.68  2.48 SEQ ID 110.51  7.81 SEQ ID180.00  7.07 SEQ ID 131.68  8.77 SEQ ID 121.07  5.86 NO: 20 NO: 50 NO:85 NO: 118 NO: 148 SEQ ID 116.67  4.01 SEQ ID 158.00 13.73 SEQ ID 267.0316.40 SEQ ID 121.04  6.30 GFP 100.00  6.55 NO: 21 NO: 51 NO: 87 NO: 119SEQ ID 131.67  8.00 SEQ ID 119.42  8.70 SEQ ID 173.33  4.58 SEQ ID104.85  7.51 NO: 22 NO: 53 NO: 89 NO: 120 SEQ ID 124.29  6.45 SEQ ID145.00 11.92 SEQ ID 135.00  9.29 SEQ ID 113.85  6.36 NO: 23/24 NO: 54NO: 90 NO:121 DR-performance (leaf area) under Drought shown in % of GFPexpressing plants SD-value shown ± standard deviation

As shown in Table 5, all plants expressing the tested genes identifiedby the method of the present invention revealed increased leaf area byabout 15% to about 90% under drought conditions as compared to plantsexpressing the negative control gene (GFP). These results demonstratethat the method of the present invention provides novel genes conferringimproved drought tolerance in plants.

Reference is now made to Table 6 presenting results of droughtexperiments conducted with T2 Arabidopsis plants re-cloned with therelevant Seq. IDs. Different Seq. IDs were re-cloned and re-transformedinto Arabidopsis plants generating several independent events(represented by E1-3 in Table 6). Plants were grown in soil incontrolled greenhouses and tested for drought tolerance under theconditions mentioned above. During their growth, images were taken andimage analysis was applied, converting the images into leaf area perplant. Results are shown in Table 6 as percentage of GFP expressingplants that served as a negative control during the drought phase.

TABLE 6 Results of drought experiments conducted with T2 Arabidopsisplants re-cloned with the relevant Seq. IDs Seq ID DR RC E1 E1 ± SD DRRC E2 E2 ± SD DR RC E3 E3 ± SD SEQ ID NO: 2 114.05 12.00 126.33 6.2794.42 14.75 SEQ ID NO: 7 125.74 7.50 118.43 12.40 82.39 17.20 SEQ ID NO:8 126.96 10.73 110.07 13.09 132.74 5.34 SEQ ID NO: 9 159.34 19.75 151.9927.05 113.97 18.65 SEQ ID NO: 10 185.23 19.29 165.97 30.99 90.04 9.27SEQ ID NO: 11 116.91 9.54 106.90 10.41 106.32 10.87 SEQ ID NO: 12 178.8024.09 107.57 14.72 157.66 15.22 SEQ ID NO: 14 162.78 14.10 151.93 9.90123.68 10.10 SEQ ID NO: 16 144.23 8.42 141.32 7.03 127.03 8.31 SEQ IDNO: 18 176.30 26.57 126.24 11.63 138.53 23.03 SEQ ID NO: 22 113.00 12.14109.38 9.14 105.16 12.38 SEQ ID NO: 25 150.56 7.57 153.02 9.91 120.2513.63 SEQ ID NO: 28 193.32 28.79 SEQ ID NO: 30 123.33 11.83 113.97 8.18112.53 16.34 SEQ ID NO: 33 141.20 10.90 127.98 13.30 112.63 11.50 SEQ IDNO: 34 167.25 12.60 150.19 13.30 138.48 10.20 SEQ ID NO: 41 160.43 11.60153.92 14.10 112.83 10.80 SEQ ID NO: 43 229.50 18.12 136.33 32.37 106.8326.53 SEQ ID NO: 51 178.07 13.10 170.57 14.60 146.17 11.20 SEQ ID NO: 54169.39 15.50 131.72 11.30 120.10 16.70 SEQ ID NO: 55 126.72 16.39 122.4818.62 111.94 17.92 SEQ ID NO: 56/57 138.08 8.64 134.76 9.21 127.74 10.65SEQ ID NO: 58 115.36 11.52 117.79 13.24 93.16 11.94 SEQ ID NO: 60 151.9012.80 137.24 11.90 93.80 5.60 SEQ ID NO: 61 140.14 12.10 116.31 14.70114.09 10.30 SEQ ID NO: 74/75/76 175.07 13.50 160.92 12.30 105.95 11.30SEQ ID NO: 77 210.21 18.03 174.80 18.44 160.93 29.97 SEQ ID NO: 78182.00 15.30 175.52 16.80 115.61 11.10 SEQ ID NO: 85 132.73 10.80 119.8611.50 114.46 9.90 SEQ ID NO: 89 167.95 21.26 154.64 21.46 142.21 29.65SEQ ID NO: 90 141.50 24.45 137.53 17.22 110.29 32.15 SEQ ID NO: 91/92219.30 29.16 192.51 22.47 92.77 20.90 SEQ ID NO: 93 127.73 16.50 122.9911.32 119.54 17.08 SEQ ID NO: 94 123.64 13.85 120.32 9.86 107.77 15.59SEQ ID NO: 95 129.53 9.05 108.36 9.42 98.43 14.09 SEQ ID NO: 101 161.6814.10 141.20 11.30 134.68 13.60 SEQ ID NO: 105 204.51 27.93 188.14 5.31156.19 17.89 SEQ ID NO: 106 153.33 12.60 143.91 10.80 130.47 11.50 SEQID NO: 109 141.18 14.20 134.15 11.60 124.80 10.30 SEQ ID NO: 110 118.6610.30 113.58 8.40 104.01 7.60 SEQ ID NO: 111/112 228.16 35.62 202.4318.73 132.98 18.32 SEQ ID NO: 113 158.59 24.54 155.03 21.36 135.44 17.44SEQ ID NO: 126 185.07 13.40 147.37 16.20 131.05 10.80 DR-performance(leaf area) under drought shown as % of GFP expressing plants RCE1-3-performance with re-cloned relevant Seq. ID event 1-3 SD-valueshown ± standard deviation

As shown in Table 6, plants expressing the re-cloned genes identified bythe method of the present invention presented enhanced leaf area ascompared to plats expressing the negative control gene, in Arabidopsisplants subjected to drought conditions.

Reference is now made to Table 7 presenting results of droughtexperiments conducted with T2 tobacco plants. Different genes identifiedby the present invention were re-cloned and transformed into tobaccoplants generating several independent events (represented by E1-3 inTable 7). Plants were grown in soil in controlled greenhouses and testedfor drought tolerance under the conditions mentioned above. At the endof the experiment plant shoots fresh weight, leaves number, length ofmain branch and weight of main branch were evaluated. Results are shownin Table 7 as percentage of wild type (WT) plants that served as anegative control.

TABLE 7 Results of drought experiments conducted with T2 Tobacco plantsSeq ID FW FW ± SD LN LN ± SD BFW BFW ± SD BL BL ± SD SEQ ID NO: 1 E1104.71 11.18 73.58 11.63 106.78 14.75 122.73 9.25 SEQ ID NO: 1 E2 97.282.18 67.92 4.76 86.18 5.10 97.27 8.86 SEQ ID NO: 1 E3 99.22 11.21 64.157.70 116.72 6.30 118.18 8.90 SEQ ID NO: 2 E1 122.23 2.70 116.76 4.84115.42 3.47 97.33 4.87 SEQ ID NO: 2 E2 119.11 5.62 101.62 3.97 122.992.70 114.84 2.98 SEQ ID NO: 2 E3 116.69 9.00 111.35 4.58 117.50 14.45102.67 9.91 SEQ ID NO: 15 E1 111.60 4.18 98.38 7.58 113.65 4.39 102.084.89 SEQ ID NO: 15 E2 121.87 2.88 118.92 2.53 122.25 3.70 100.59 3.71SEQ ID NO: 15 E3 113.93 5.39 116.76 6.42 103.32 7.79 95.25 9.41 SEQ IDNO: 44 E1 124.04 4.23 118.92 6.69 130.31 5.23 94.66 6.12 SEQ ID NO: 44E2 121.17 8.34 108.11 3.54 128.45 14.00 112.02 8.14 SEQ ID NO: 44 E3113.80 10.36 117.84 7.28 118.83 9.15 90.80 8.89 SEQ ID NO: 55 E1 120.523.43 123.24 5.53 122.56 5.65 102.08 5.33 SEQ ID NO: 55 E2 117.85 8.35113.51 3.42 121.35 11.26 95.55 7.40 SEQ ID NO: 55 E3 123.13 5.02 111.359.31 127.92 8.75 110.09 7.81 SEQ ID NO: 56/57 E1 101.58 5.17 75.47 8.9280.26 23.40 109.55 6.45 SEQ ID NO: 56/57 E2 106.93 9.10 79.25 8.50101.30 10.14 103.64 7.22 SEQ ID NO: 56/57 E3 98.16 10.90 75.47 8.9281.97 10.54 100.91 15.84 SEQ ID NO: 142 E1 110.50 5.07 94.34 15.46109.37 7.31 116.82 16.89 SEQ ID NO: 142 E2 119.83 5.07 94.34 1.98 105.755.15 118.64 19.00 SEQ ID NO: 142 E3 114.89 2.74 98.11 6.86 101.90 5.9495.45 19.44 WT 100.00 2.43 100.00 1.67 100.00 9.84 100.00 11.65 FW-freshweight measured in grams LN-leaf number BFW-branch fresh weight measuredin grams BL-main branch length measured in cm SD-value shown +/−standard deviation as % of measured trait E1-3-different independentevents

The results presented in Table 7 show that most of the genes identifiedby the present invention confer improved tolerance to drought conditionsin Tobacco plants, as shown by the tested parameters (e.g. fresh weight,leaf number, branch fresh weight, branch length) as compared to negativecontrol plants.

Reference is now made to Table 8 presenting results of salinityexperiments of transgenic tobacco plants as compared to control WTplants. Different tobacco lines expressing various genes identified bythe method of the present invention (see Table 4), were germinated insoil. Seven days post germination; plants were irrigated with fertilizedwater containing 400 mM NaCl. Leaf images were taken 14 days afterirrigation with salt and analyzed for leaf area for the differentindependent events. Results are shown in Table 8 as percentage leaf areadifference from WT plants.

TABLE 8 Results of salinity experiments on tobacco plants Seq ID HST ±SD SEQ ID NO: 1 225.12 12.65 SEQ ID NO: 2 240.63 28.91 SEQ ID NO: 15505.52 17.57 SEQ ID NO: 44 767.46 7.48 SEQ ID NO: 55 206.71 26.27 SEQ IDNO: 56/57 286.19 4.86 SEQ ID NO: 70/71 1366.07 4.70 SEQ ID NO: 142318.54 29.75 WT 100.00 13.22 HST- high salinity tolerance shown as %difference of leaf area as compared to WT SD-value shown +/− standarddeviation between 4 independent events

The results of Table 8 clearly show that plants expressing the novelsalinity tolerance genes identified by the present invention revealedsignificantly higher leaf area as compared to WT control plants.

Reference is now made to Table 9 presenting salinity experimentsconducted on Arabidopsis plants expressing novel genes having Seq. IDsas indicated. Ten plants per event per pot were grown in soil incontrolled greenhouse. After germination, all pots with plants wereirrigated by submerging them with 100 mM NaCl. The results of Table 9represent average data of 4 different events per Seq. ID and wild typeplants (WT).

TABLE 9 Results of salinity experiments conducted on Arabidopsis plantsexpressing novel identified genes Flower & Pod Seq ID production FP ± SDChlorosis Chlor ± SD SEQ ID NO: 1 2.50 0.50 4.17 0.00 SEQ ID NO: 2 3.750.48 4.58 0.00 SEQ ID NO: 5 1.00 0.41 2.33 1.00 SEQ ID NO: 6 3.25 0.254.42 0.00 SEQ ID NO: 7 3.50 0.50 4.42 0.25 SEQ ID NO: 8 4.00 0.41 4.670.00 SEQ ID NO: 9 3.25 0.25 4.33 0.25 SEQ ID NO: 10 2.25 0.48 3.42 0.41SEQ ID NO: 11 1.50 0.50 3.00 0.50 SEQ ID NO: 12 2.75 0.63 3.83 0.63 SEQID NO: 13 2.75 0.25 3.58 0.00 SEQ ID NO: 16 2.75 0.63 4.08 0.00 SEQ IDNO: 18 1.50 0.29 2.92 0.25 SEQ ID NO: 22 2.50 0.87 3.33 0.41 SEQ ID NO:23/24 3.50 0.29 4.50 0.00 SEQ ID NO: 25 2.25 0.75 3.08 0.29 SEQ ID NO:26/27 1.75 0.48 3.00 0.25 SEQ ID NO: 29 2.50 0.29 3.83 0.25 SEQ ID NO:30 3.25 0.48 4.25 0.29 SEQ ID NO: 30 3.00 0.71 4.33 0.00 SEQ ID NO: 332.50 0.87 3.42 0.25 SEQ ID NO: 94 2.50 0.29 3.67 0.29 SEQ ID NO: 40 1.250.48 2.33 1.25 SEQ ID NO: 43 2.00 0.41 3.75 0.25 SEQ ID NO: 44 3.50 0.294.42 0.25 SEQ ID NO: 55 2.00 0.41 3.67 0.00 SEQ ID NO: 56/57 2.50 0.293.58 0.25 SEQ ID NO: 58 1.75 0.85 2.67 1.04 SEQ ID NO: 59 3.00 0.41 3.750.48 SEQ ID NO: 70/71 1.75 0.63 2.67 1.00 SEQ ID NO: 77 2.00 0.00 3.500.29 SEQ ID NO: 89 1.75 0.63 2.50 1.11 SEQ ID NO: 90 3.00 0.71 3.83 0.25SEQ ID NO: 91/92 1.00 0.00 3.17 0.58 SEQ ID NO: 93 2.75 0.25 4.17 0.25SEQ ID NO: 95 3.75 0.25 4.50 0.25 SEQ ID NO: 99 2.00 0.41 3.00 0.25 SEQID NO: 103/104 2.00 0.00 3.67 0.29 SEQ ID NO: 106 1.00 0.00 2.25 0.29SEQ ID NO: 107 1.75 0.25 3.00 0.25 SEQ ID NO: 110 1.75 0.25 2.83 0.25SEQ ID NO: 111/112 1.00 0.00 3.08 0.25 SEQ ID NO: 113 1.25 0.75 1.831.44 SEQ ID NO: 118 2.50 0.29 3.25 0.50 SEQ ID NO: 119 2.25 1.03 2.751.19 SEQ ID NO: 120 2.00 0.41 3.08 0.29 SEQ ID NO: 124 1.75 0.25 3.000.00 HRD 1.25 0.25 2.83 0.48 WT 1.00 0.00 1.83 0.55 FP-Flowers and podsproduction 1-No Flowers 2-Few flowers formation with short floweringstems 3-Some flower formation almost no pods 4-Flowers and pods formingChlorosis-Chlorosis and damage to leaves 1-Completely dry leaves 2-Dryleaf edges 3-Yellow 4-Some Yellow 5-Green ± SD-standard deviation

As shown in Table 9, plants expressing genes identified by the method ofthe present invention as conferring salinity tolerance, demonstratedsignificantly higher flowers and pods yield and significantly reducedchlorosis and damage effects to the leaves as compared to WT controlplants subjected to the same salinity stress conditions.

To conclude, the experimental results presented above clearlydemonstrate that by the unique method of the present invention, highlyvaluable stress tolerance (e.g. drought, salinity) genes in plants canbe identified. The newly identified genes confer improved tolerance orresistance to the preselected stress in plants in various importantparameters such as leaf area, turgor pressure, aerial yield and quality,flowers and fruits yield etc. These results show that the presentinvention provides a novel screening method that identifies stresstolerance plant genes that can be expressed in desirable and importantcrops to enable their growth and enhance their yield under variousabiotic and biotic stress conditions.

REFERENCES

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1. A plant comprising a transgene encoding a non-plant polypeptidesequences having at least 90% identity to the polypeptide sequences setforth in SEQ ID NO: 177 and wherein the plant has one or more of thefollowing characteristics: improved drought resistance, increasedbiomass, increased salinity tolerance.
 2. The plant according to claim1, wherein said plant has at least one plant improving trait as comparedto a plant of the same genus lacking the transgene.
 3. The plant ofclaim 1, wherein the transgene identified has a polynucleotide sequenceshaving at least 80% identity to the polynucleotide sequences set forthin SEQ ID NO:33.
 4. The plant according to claim 1, wherein the plant isfurther characterized by improved nutrition value of the crop, improvedgrain yield, increased herbicide or chemical resistance or tolerance,increased resistance to cold, increased resistance to hear, improvedfertilizer uptake, improved fertilizer usage or any combination thereof.5. The plant according to claim 1, wherein the plant is an agriculturalcrop.